A sensitive and specific high-performance liquid chromatographic method for the determination of hydroxytolbutamide (HTB), formyltolbutamide (FTB), and carboxytolbutamide (CTB) in rat blood and urine is described. The ethanol-treated blood and urine were chromatographed on a reverse-phase column on a high-performance liquid chromatograph equipped with a ultraviolet absorbance detector. Quantitation of HTB, FTB, and CTB in blood samples containing less than 100 μg/ml of these compounds was achieved, using chlorpropamide as an internal standard. There were no interfering peaks due to other components of blood or urine.