Hydroxylations of dehydroepiandrosterone (DHEA) at the 7 alpha- and 7 beta- positions have been reported in numerous murine tissues and organs, including liver, and the responsible cytochrome P450 (P450) species await identification. Using thin layer chromatography and gas chromatography-mass spectrometry, we report identification of 7 alpha-hydroxy-DHEA and 7 beta-hydroxy-DHEA metabolites produced in mouse liver microsome digests and kinetic studies of their production with apparent KM values of 3.19 +/- 0.292 microM and 2.82 +/- 0.241 microM for 7 alpha- and 7 beta-hydroxylation, respectively. Investigation of P450 inhibitor and of steroid hormone effects on both 7 alpha- and 7 beta-hydroxylation of DHEA showed that, 1) different P450s were involved in 7 alpha- and 7 beta-hydroxylation of DHEA because metyrapone inhibited solely 7 alpha-hydroxylation, 2) P450 2D6, 2B1, and 2B11 were not responsible for 7 alpha- and 7 beta-hydroxylation of DHEA because respective specific inhibitors quinidine and chloramphenicol triggered no inhibition, 3) aside from P450 7b, P450 1A1, and 1A2 may be responsible for a fraction of DHEA 7 alpha- and 7 beta-hydroxylation because alpha-naphthoflavone and furafylline, which inhibit specifically P450 1A1 and 1A2, decreased the 7 alpha- and 7 beta-hydroxylation partly, 4) comparison of these findings with those obtained with brain microsomes suggested that tissue-specific P450 species are responsible for the 7 alpha- and 7 beta-hydroxylation of DHEA, 5) 7 alpha-hydroxylation of DHEA may be shared with other 3 beta-hydroxysteroids, such as 3 beta-hydroxy-5 alpha-androstan-17-one, 5-androstene-3 beta,17 beta-diol and pregnenolone, which acted in a noncompetitive manner. Taken together, these findings will be of use for identification of the P450 species responsible for 7 alpha- and 7 beta-hydroxylation of DHEA and for studies of their activities in liver.