In an enzymatic assay for chloramphenicol, chloramphenicol acetyltransferase partially purified by affinity chromatography was used; [3H]acetyl Co A served as a substrate. The purified enzyme was sensitive to p-hydroxymercuribenzoate but insensitive to 5,5''-dithio-bis-2-nitrobenzoic acid. The Km was 10.5 .mu.M. The operating range of the enzymatic assay was 0-30 .mu.g/ml. The coefficient of variation was 1.9% at a concentration of 10.mu.g/ml. The method correlated well with a microbiological agar-paper disk method for mock unknown sera (r = 0.997) and serum or urine specimens (r = 0.993). The enzymatic assay was unaffected by the presence of any of 12 other antibiotics in tested serum.