We describe a flame-ionization gas-chromatographic procedure for determination of the potentially toxic antibiotic, chloramphenical, in serum. The serum (500 mul) is extracted into ethyl acetate and nonpolar impurities are subsequently partitioned into hexane. The drug is chromatographed as its bis-trimethylsilyl derivative, with the analog thiamphenicol as the internal standard. Within-run precision (CV) is 4.4% at a serum concentration of 41 mg/liter and 9.2% at a concentration of 5 mg/liter. Over a six-month period, the run-to-run variation was 5.1% at 40 mg/liter (n = 24). Results by the gas-chromatographic method compared well with those by an established colorimetric procedure; mean concentrations for the comparison samples in the two procedures were 18.4 mg/liter and 17.6 mg/liter, respectively (n = 27), with a coefficient of correlation of 0.998. The gas-chromatographic method is more precise and specific than classical microbiological procedures and is suitable for routine therapeutic monitoring of serum chloramphenicol concentrations.