In vitrostudies with human liver preparations suggest that the metabolism of hexobarbital involves CYP2CMP— the determinant of theS-mephenytoin 4‘-hydroxylation polymorphism, but no invivoevidence of interphenotypic differences exist. The pharmacokinetics and urinary excretion of hexobarbital and its metabolites were, therefore, investigated following oral administration of a differentially labelled pseudoracemate that allowed determination of the fate of the individual enantiomers. Studies were undertaken in 10 Caucasian and nine Chinese healthy subjects known to be either extensive (EM) or poor (PM), metabolizers of mephenytoin. No inter-racial differences were observed in any of the measured parameters within a given phenotype. However, pronounced stereoselectivity in disposition was noted in EMs withR-( – )-hexobarbital's oral clearance being five- to six-fold greater than that for theS-( + )-enantiomer. By contrast, theS-( + )-isomer was eliminated twice as fast asR-( — ) hexobarbital in PMs and, in addition, the oral clearances of both enantiomers were significantly reduced compared with their values in EMs. Formation of 3’-hydroxy- and 3‘-ketohexobarbital and 1,5-dimethyIbarbituric acid were the major identified routes of metabolism for each enantiomer in both phenotypes. Furthermore, these pathways were found to co-segregate with the mephenytoin polymorphism and in EMs they were primarily responsible for the observed stereoselectivity in disposition. These findings, therefore, confirm the stereoselectivity in hexobarbital's disposition in humans and identify the major pathways of metabolism involved. Additionally, the results indicate that CYP2CMPis a major determinant of the invivometabolism of both of hexobarbital's enantiomers but especially that of theR-( — )-enantiomer.