Radioimmunoassays were developed for R- and S-pentobarbital. The optical isomers of pentobarbital were individually alkylated to N-crotonic acid analogs that were coupled to bovine serum albumin. Immunization of rabbits with the conjugates, which were enantiomerically pure at the asymmetric' carbon of the pentobarbital moiety, led to formation of antisera that selectively bound the predicted enantiomer. In displacement studies with enantiomerically pure radioligands, the opposite enantiomer showed 1.0 to 1.4% cross-reaction. Similar selective binding was observed for enantiomers of secobarbital, thiopental and thiamylal. Assays were developed and used to determine enantiomer pharmacokinetics in rabbits and humans given racemic pentobarbital. In rabbits, difference in clearance of the two isomers was minimal, the result of a slightly larger volume of distribution of the R-enantiomer combined with a slightly higher value of the elimination rate constant beta for the S-enantiomer. In humans, the volume of distribution was 12% greater for the R-enantiomer, but the value of beta was 14% higher for this isomer as well. Thus, the median clearance of the S-enantiomer (1.96 liters/h) was 25% less than that of the R-isomer (2.58 liters/h). The S-enantiomer was also more strongly protein bound in plasma (73.5% vs 63.4% for the R-enantiomer), which is consistent with its structural congruence to S-warfarin, S-phenprocoumon and S-glifumide.