In many human and murine tissues, both pregnenolone and dehydroepiandrosterone are hydroxylated at the 7alpha and 7beta positions by a cytochrome P450-containing microsomal complex. The 7alpha- and 7beta-hydroxysteroids produced were shown to activate an immune response in mice. Based upon identification by crystallization to constant specific activity and gas chromatography-mass spectrometry analysis, we ascertained that a yeast-expressed human cytochrome P450-1A1 was able to 7beta-hydroxylate pregnenolone (K(M) from 3.2 +/- 0.5 to 4.1 +/- 0.4 microM, turnover number from 117 +/- 15 to 135 +/- 13 pmol/min/nmol of cytochrome P450-1A1). The other human cytochromes P450 tested did not produce identifiable quantities of 7alpha- or 7beta-hydroxylated derivatives of pregnenolone or dehydroepiandrosterone. These findings indicate that cytochrome P450-1A1 involvement in the 7beta-hydroxylation of pregnenolone may contribute to the production of the 7-hydroxylated steroids necessary for activation of the immune defences.