The aim of this paper was to validate an analytical method for the simultaneous determination of PTX and its active metabolite (-)-(R)-M1 in rat serum and some tissues using a high-performance liquid chromatography method with ultraviolet detection (HPLC-UV). The specificity, linearity, precision, accuracy, recovery, lower limit of detection, lower limit of quantification and stability study were successively conducted according to GLP procedures. HPLC separation of all compounds was carried out on a normal-phase ChiralPak AD column (250 mm x 4.6 mm i.d., 5 mm), using, as a mobile phase, a mixture of hexane and 2-propanol (84:16, v/v) containing 0.01% of diethylamine with a flow rate of 1.5 mL x min(-1). The calibration curves from all studied matrices were linear across the concentration range from 0.01 to 100 mg x mL(-1) with a lower limit of quantification of 0.01 microg x mL(-1) for all analytes. The application of the assay to a pilot pharmacokinetic study and tissue distribution of the compounds in rats after intraperitoneal dosing of 50 mg x kg(-1) of PTX was described. Significant (p<0.05) differences between serum and tissue levels of PTX, (-)-(R)-M1 and (+)-(S)-M1 were observed.