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This report describes a rapid and sensitive analytical method for the quantification of the neuroactive steroids alphaxalone and pregnanolone in rat plasma using derivatization with dansyl hydrazine as fluorescent label. The method involves protein precipitation, alkaline derivatization and extraction of the compounds and internal standard pregnenolone with dichloromethane, followed by isocratic reversed-phase high-performance liquid chromatography on a 3-microm Microsphere C18 column with fluorescence detection at wavelengths 332 nm and 516 nm for excitation and emission, respectively. The mobile phase consists of a mixture of 25 mM acetate buffer (pH 3.7)-acetonitrile (45:55, v/v for alphaxalone and 40:60, v/v for pregnanolone) with a flow-rate of 1 ml/min. The total run time was approximately 35 min. In the concentration range of 0.010-10 microg ml(-1), the intra- and inter-assay coefficients of variation were less than 17% for both methods. In 50 microl plasma samples the corresponding limits of detection were 10 ng ml(-1) (signal-to-noise ratio=3). The utility of the analytical method was established by analyzing plasma samples from rats, which had received an intravenous administration of 5 mg kg(-1) alphaxalone or pregnanolone. Values for clearance, volume of distribution at steady state and terminal half life were 71.9 ml min(-1) kg(-1), 814 mg kg(-1) and 13.5 min for alphaxalone and 69.2 ml min(-1) kg(-1), 1,638 ml kg(-1) and 27.8 min for pregnanolone, respectively. Due to its simplicity and sensitivity this method can be used on a routine basis for pharmacokinetic analysis of neuroactive steroids.