We describe a method for measuring chloramphenicol by high-performance liquid chromatography. The assay involves a single extraction of the biological sample with ether, evaporation of the extract, and chromatography of the residue, redissolved in methanol. A reversed-phase column with an eluting solvent of methanol/water (30/70 by vol) is used. Chloramphenicol is eluted from the column in about 4 min and is well separated from the internal standard (mephenesin), which is eluted at 5.5 min. Absorption of the effluent at 278 nm is monitored and measured. As little as 0.1 microgram of the antibiotic can be analyzed after its extraction from a 0.1-ml sample. The method is suitable for rapid and specific analysis for the drug in plasma, urine, cerebrospinal fluid, and other biological fluids.