Hydroxylations of pregnenolone (PREG) at the 7α- and 7β- 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 and crystallization to constant specific activity, we report identification of 7α-hydroxy-PREG and 7β-hydroxy-PREG metabolites produced in mouse liver microsomes and kinetic studies of their production with apparent K M values of 2.45 ± 0.124 μM and 3.41 ± 0.236 μM for 7α- and 7β-hydroxylation, respectively. Investigation of P450 inhibitors and of steroid hormone effects on both 7α- and 7β-hydroxylation of PREG showed that 1) different P450 were involved because metyrapone and antipyrine inhibited solely 7α- and 7β-hydroxylation, respectively; 2) P450 1A2, 2D6, 2B1, and 2B11 were not responsible for 7α and 7β-hydroxylation of PREG because respective specific inhibitors furafylline, quinidine, and chloramphenicol triggered no inhibition; 3) P450 1A1 was responsible for only part of the 7β-hydroxylation of PREG because α-naphthoflavone, which inhibits specifically P450 1A1, did not suppress entirely 7β-hydroxylation while ketoconazole, antipyrine, and metyrapone extensively decreased the 7β-hydroxylation; 4) comparison of these findings with those obtained with brain microsomes suggests that tissue-specific P450 species are responsible for the 7α- and 7β-hydroxylation of PREG; and 5) 7α-hydroxylation of PREG may be shared with other 3β-hydroxysteroids such as isoandrosterone, 5-androstene-3β,17β-diol, and dehydroepiandrosterone, which acted in a competitive manner. Taken together, these findings will be of use for identification of the P450 species responsible for 7α- and 7β-hydroxylation of PREG and for studies of their activities in liver and other organs.