Mouse steroid 15 alpha-hydroxylase P-450 2a-4 is restricted in its substrate specificity to the delta 4, 3-ketone steroids such as androstenedione. As a result, the P-450 exhibits little hydroxylase activity toward delta 5, 3-hydroxysteroids including dehydroepiandrosterone (DHEA). A single amino acid mutation of Ala at position 117 to Val, however, is enough to confer a high DHEA hydroxylase activity to P-450 2a-4 with 7 alpha-OH DHEA as one of the two major hydroxylated metabolites. Mouse coumarin 7-hydroxylase P-450 2a-5 contains Val at position 117, but it exhibits very low DHEA hydroxylase activity. P-450 2a-5 acquires high DHEA hydroxylase activity, however, by a mutation of Phe-209 to Asn. Moreover, the mutant P-450 2a-5 loses its activity when Val is replaced by Ala at position 117. The residue at position 117, therefore, plays the principal role in the determination of the DHEA hydroxylase activity of the P-450s. Conversely, mutations at residue 117 have little effect on the androstenedione hydroxylase activities of the P-450s. Further modeling of the DHEA binding orientation in the substrate-heme pocket of bacterial P-450cam (Iwasaki, M., Darden, T., Pedersen, L., Davis, D. G., Juvonen, R. O., Sueyoshi, T., and Negishi, M. (1993) J. Biol. Chem. 268, 759-762) provides support for the hypothesis that the type of residue at position 117 determines the steroid-substrate specificity of the P-450 depending on the substituent at the C3 position of steroid molecule.