CGP 43371 (compound I), a mono-pivaloyl oxazole derivative of a 3-piperazino-rifamycin, has been in clinical trials as a potential hypocholesterolemic agent. A reversed-phase high-performance liquid chromatographic (HPLC) assay was developed using a C18 column and a gradient solvent system of methanol-0.1 M sodium acetate, pH 4.5, at a flow-rate of 1 ml/min. The compound and internal standard (rifampicin) were detected by their ultraviolet absorption at 254 nm. Isolation of the compounds from plasma and liver homogenates was accomplished by precipitation of proteins with acetonitrile, followed by evaporation under nitrogen and reconstitution in methanol. Bile, lymph and urine were injected onto the HPLC column without pretreatment. Calibration curves were linear (r > 0.999) over the concentration range 0.25-20.0 micrograms/ml. The assay procedure was also applicable to other rifamycin derivatives and was able to distinguish between molecular species containing small differences in functionality.