A new, highly sensitive method for quantitative separation and determination of capsaicin and dihydrocapsaicin by high-performance liquid chromatography with electrochemical detection is elaborated. The method employs a Cosmosil 5Ph column eluted with 100 mM potassium dihydrogenphosphate containing 45% acetonitrile (pH 5.0) at a flow-rate of 1.0 ml/min, and an electrochemical detector (at a potential of +750 mV versus the Ag/AgCl electrode). The detection limits for both capsaicin and dihydrocapsaicin were 12 pg (39 fmol) at a signal-to-noise ratio of 3:1. By applying this method, the biological half-life of capsaicin in the rat was investigated.