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General reference information for researchers and lab staff, not medical or safety advice for any individual. Always follow the supplier safety data sheet for the lot in hand and your institution’s chemical hygiene plan.
Short answer
Cycloheximide (C15H23NO4, molecular weight 281.35 per PubChem) is a small-molecule inhibitor of protein synthesis in eukaryotic cells. It blocks translation elongation, which is why labs use it in cycloheximide chase assays to follow how fast a protein disappears once new synthesis is stopped. It is also a hazardous chemical: PubChem’s aggregated GHS data include H300 (fatal if swallowed) and reproductive and genetic toxicity statements.
What is cycloheximide? Definition and scope
PubChem (CID 6197, read 2026-11-07) describes cycloheximide as a dicarboximide, an antibiotic produced by the bacterium Streptomyces griseus, with the role of a protein synthesis inhibitor. Its CAS number is 66-81-9. Because it acts on eukaryotic ribosomes, it is used as a research reagent and agricultural fungicide, not as a treatment for infections; PubChem’s EPA-derived description lists its uses as a fungicide and an anticancer drug.
Cycloheximide mechanism of action
Cell Signaling Technology’s datasheet #2112 (read 2026-11-07) calls it a protein synthesis inhibitor in eukaryotes that inhibits translation elongation by binding the E-site of the 60S ribosomal subunit and interfering with deacetylated tRNA, while noting the precise mechanism has yet to be fully elucidated. It cites Schneider-Poetsch et al., Nat Chem Biol 2010; 6:209, and Klinge et al., Science 2011; 334:941. The same datasheet reports that it induces cell death in T cells through a FADD-dependent mechanism and acts synergistically with TNF, so effects on a cell are not limited to silent translation arrest.
How it is used in practice
The best-known use is the cycloheximide chase: after translation is blocked, remaining protein is measured over time (commonly by western blotting) to estimate protein half-life. Because the drug itself can trigger cell death, as above, treatment length is a design decision to document rather than a fixed rule.
Cycloheximide stock solution and working concentration
The CST datasheet reports that the product is supplied lyophilized, that a 10 mg/ml stock corresponds to 50 mg in 5 ml of DMSO or ethanol, and that it is typically used at 5-50 µg/ml for 4-24 hours, with working concentration and duration varying with the desired effect. Other suppliers and papers use other ranges, so record the concentration, solvent and exposure time you actually used.
How long can you treat cells with cycloheximide?
CST gives 4-24 hours as the typical range and states that the length varies with the desired effect. Since the compound can induce cell death and synergize with TNF, longer exposures need a viability check appropriate to the cell type; that choice belongs in the experimental protocol, not in a generic rule.
Cycloheximide solubility: does it dissolve in water?
Yes, to a degree, but sources disagree on the number. This is the comparison the vendor pages do not make:
| Source (as read 2026-11-07) | Reported solubility |
|---|---|
| CST datasheet #2112 | H2O 20 mg/ml; DMSO 25 mg/ml; also soluble in ethanol and methanol |
| NTP 1992, cited in PubChem | 10 to 50 mg/mL in water at 68 F |
| Merck Index 1996 and ICSC, cited in PubChem | 2.1 g/100 mL in water at 2 C |
One further water-solubility entry in PubChem (2.1e-4 mg/L) is inconsistent with all of these and appears to be a unit error, so it should not be used. Practically: water solubility is on the order of tens of mg/ml, and the figure depends on the source and temperature. Use the value on your supplier’s datasheet for your lot.
Is cycloheximide toxic to humans? SDS and hazard classes
Yes, it is classified as highly hazardous. PubChem’s aggregated GHS classification (varies by notifier) includes Danger; H300 fatal if swallowed; H315 skin irritation; H319 eye irritation; H341 suspected of causing genetic defects; H360 and H360D may damage fertility or the unborn child; and H411 toxic to aquatic life with long-lasting effects. Classifications differ across notifiers: PubChem also records an NITE (FY2018) entry of “Not Classified”. That spread is the reason to read the SDS from your own supplier (see how to read a safety data sheet) rather than copy hazard statements from a web summary, and to map labels with the GHS classification guide.
Where it matters for labs: a handling and documentation checklist
These items come from the CST datasheet’s safety statements and PubChem’s hazard data:
- Treat it as suspected of causing genetic defects and possibly adverse reproductive effects; CST advises avoiding contact during pregnancy and nursing.
- Wear personal protective equipment as the SDS specifies.
- Do not empty it into drains (CST); PubChem lists an aquatic hazard statement (H411).
- Keep the supplier SDS for each lot with the lab’s chemical inventory, in line with the Hazard Communication Standard in laboratories.
- Record solvent, stock concentration, working concentration and exposure time in the experiment record.
Common misunderstandings
- “It is an antibiotic, so it is safe in cell culture.” PubChem does call it an antibiotic produced by Streptomyces griseus, but it inhibits eukaryotic protein synthesis, which is why it is toxic to human cells and used as a research tool.
- “It only stops protein synthesis.” CST notes FADD-dependent cell death in T cells and synergy with TNF.
- “Every source gives one solubility.” See the table above.
- “The hazard class is settled.” Notifier classifications differ; the SDS for your lot governs.
Frequently asked questions
What is cycloheximide?
A small-molecule inhibitor of eukaryotic translation elongation, produced by Streptomyces griseus, used mainly as a research reagent.
Is cycloheximide an antibiotic?
PubChem describes it as an antibiotic produced by Streptomyces griseus, but it acts on eukaryotic ribosomes and is a toxic research chemical rather than a treatment for infection.
Is cycloheximide toxic to humans?
PubChem’s aggregated GHS data include H300 (fatal if swallowed) and reproductive and genetic toxicity statements; follow the supplier SDS.
Does cycloheximide dissolve in water?
Yes; reported values range from about 10 to 50 mg/mL (NTP) and 20 mg/ml (CST) to 2.1 g/100 mL at 2 C (Merck Index), depending on source and temperature.
What is the cycloheximide molecular weight?
281.35 for C15H23NO4 (PubChem CID 6197, retrieved 2026-11-07).
How long can you treat cells with cycloheximide?
CST reports typical use of 4-24 hours at 5-50 µg/ml, varying with the effect sought.
Sources
- PubChem, Cycloheximide, CID 6197, pubchem.ncbi.nlm.nih.gov/compound/6197, read 2026-11-07.
- Cell Signaling Technology, Cycloheximide #2112 datasheet, media.cellsignal.com/pdf/2112.pdf, read 2026-11-07.
Related: Annexin V / PI apoptosis assay setup.
