A liquid-chromatographic assay of 5-fluorouracil in plasma is described. Advantages of this procedure over gas chromatography are the simpler extraction procedure, elimination of the need for a derivitization step with silylating agents, and a 20-fold greater sensitivity. The minimum detectable concentration of 5-fluorouracil in plasma is 25 microgram/L. The enhanced sensitivity enabled measurement of the concentrations of 5-fluorouracil found in plasms of patients receiving continuous intravenous infusions of the drug; such concentrations are generally unmeasurable by gas-chromatographic methods. We compared liquid-chromatographic and gas-chromatographic measurements on 36 plasma samples obtained from patients after rapid intravenous injection of 5-fluorouracil. The resulting correlation coefficient was 0.97, with a regression slope of 0.94. The liquid-chromatographic method is free of interference from other cytotoxic agents, anti-emetics, and folate derivatives that are frequently combined with 5-fluorouracil therapy.