A reversed-phase high-performance liquid chromatographic method for the determination of sinefungin, a new antiprotozoal drug, in rat plasma has been developed and validated. Sample preparation was performed at 4 degrees C by deproteinization with acetonitrile. Vidarabine was used as an internal standard. Both sinefungin and vidarabine were separated on a C18 column with a mobile phase of ammonium dihydrogenphosphate-acetonitrile (95:5, v/v) and detected by ultraviolet absorbance at 260 nm. Recoveries of sinefungin from plasma were 75 +/- 3.2% and 81 +/- 4.8% following dosage at concentrations of 10 micrograms/ml and 30 micrograms/ml, respectively. Using 250 microliters of rat plasma, the limit of quantitation was 1 microgram/ml sinefungin, and the assay was linear from 1 to 30 micrograms/ml. This method appears sensitive enough to be used in further pharmacokinetic studies of sinefungin in animal models.