We report a 'high-performance' liquid-chromatographic method for measuring 5-fluorocytosine in plasma and cerebrospinal fluid. After deproteinization with trichloroacetic acid, the supernates are chromatographed on a reversed-phase (C18) column. Response to concentration is linear in the range of 5 to 200 mg/L, with ultraviolet detection at 276 nm. The assay requires only 0.1 mL of plasma, is reproducible, and may be performed in less than 12 min. 5-Fluorocytosine concentrations determined by this procedure correlated well with those obtained by spectrofluorometry, although the present method is more specific with no observable interference from co-administered amphotericin B and most other commonly encountered drugs, including salicylate. This method is applicable to the routine therapeutic monitoring of pediatric and adult patients as well as to pharmacokinetic studies.