The cytochrome p450-dependent formation and subsequent interconversion of dehydroepiandrosterone (DHEA) metabolites 7 alpha-hydroxy-DHEA (7 alpha-OH-DHEA), 7 beta-hydroxy-DHEA (7 beta-OH-DHEA), and 7-oxo-DHEA was observed in human, pig, and rat liver microsomal fractions. Rat liver mitochondria and nuclei also converted DHEA to 7 alpha-OH-DHEA and 7-oxo-DHEA, but at a lower rate. With NADP(+), and less so with NAD(+), rat, pig, and human liver microsomes and rat liver mitochondria and nuclei converted 7 alpha-OH-DHEA to 7-oxo-DHEA. This reaction was inhibited by corticosterone and the 11 beta-hydroxysteroid dehydrogenase (11 betaHSD) inhibitor carbenoxolone (CBX). The conversion of 7 alpha-OH-DHEA to 7-oxo-DHEA by rat kidney occurred at higher rates with NAD(+) than with NADP(+) and was inhibited by corticosterone. With NADPH, 7-oxo-DHEA was converted to unidentified hydroxylated metabolites and low levels of 7 alpha-OH-DHEA by rat liver microsomes. In contrast, pig liver microsomal fractions reduced 7-oxo-DHEA to nearly equal amounts of 7 alpha- and 7 beta-OH-DHEA, while human fractions produced mainly 7 beta-OH-DHEA. Dehydrocorticosterone inhibited the reduction to both isomers by pig liver microsomes, but only to 7 alpha-OH-DHEA by human microsomes; CBX inhibited both reactions. Rat kidney did not reduce 7-oxo-DHEA with either NADPH or NADH. These results demonstrate that DHEA is first converted in liver to 7 alpha-OH-DHEA, which is subsequently oxidized to 7-oxo-DHEA in both liver and kidney. In liver, interconversion of 7-oxo-DHEA and 7-OH-DHEA isomers is largely catalyzed by 11 betaHSD1, while in kidney 11 betaHSD2 (NAD(+)-dependent) and 11 betaHSD3 (NADP(+)-dependent) likely catalyze the unidirectional oxidation of 7 alpha-hydroxy-DHEA to 7-oxo-DHEA. Distinct species-specific routes of metabolism of DHEA and the interconversion of its metabolites obviate extrapolation of animal studies to humans.