A simplified radioenzymatic assay for chloramphenicol was developed by eliminating the need for cumbersome extraction procedures. After the acetylation of chloramphenicol with [(14)C]acetyl coenzyme A in the presence of chloramphenicol acetyltransferase, the reaction mixture was added to a toluene-based scintillation fluid. Since (14)C-acetylated chloramphenicol is more soluble than [(14)C]acetyl coenzyme A in toluene, the radioactive product could be counted directly. The rapidity of this assay, as well as its accuracy, precision, and specificity, makes it particularly suitable for clinical use. In contrast to previous reports of enzymatic assays for chloramphenicol, we have found that results of the assay of standards prepared in serum were up to 25% higher than those of standards prepared in saline, cerebrospinal fluid, or urine.