A rapid, sensitive and enantioselective HPLC assay for the simultaneous determination of the reference 5-HT1A receptor agonists, R-(+)- and S-(-)-8-hydroxy-2-(di-n-propylamino)tetralin (R-8-OH-DPAT and S-8-OH-DPAT, respectively), in rat blood is presented. A selective extraction procedure was developed using a preliminary sample clean-up followed by isolation of R- or S-8-OH-DPAT on mixed-mode NARC-2 solid-phase columns. Separation of the enantiomers was performed by high-performance liquid chromatography using a Chiracel OD-R column. Detection was obtained using an electrochemical detector set at a voltage of 0.63 V. The mobile phase consisted of a 50 mM phosphate buffer (pH 5.5)-acetonitrile (80:20, v/v) mixture. At a flow-rate of 1 ml min(-1), the total run time was approximately 14 min. The limit of detection for R- and S-8-OH-DPAT was 0.5 ng ml(-1). In the concentration range between 50 ng ml(-1) and 1000 ng ml(-1) intra- and inter-day relative standard deviations were less than 12%. The assay was applied to a pharmacokinetic-pharmacodynamic study in rats in which decrease of body temperature was used as a measure of 5-HT1A receptor-mediated effect. Values for clearance, volume of distribution at steady state and terminal elimination rate constant were 22+/-2 ml min(-1), 1969+/-473 ml and 156+/-34 min for R-8-OH-DPAT and 16+/-1 ml min(-1), 3353+/-347 ml and 334+/-36 min for S-8-OH-DPAT, respectively. No enantiomeric interconversion was observed in vivo from R-8-OH-DPAT to S-8-OH-DPAT or vice versa.