Abstract Four double and four triple site-directed mutants of cytochrome P450 2B1 were constructed, expressed in COS cells, and assayed for androstenedione and testosterone hydroxylation. The mutants combined a Val-363 → Ala substitution with an Ile-114 → Val or Ala substitution and/or a Gly-478 → Ala or Ser substitution. Each of the individual mutations enhances androgen 15α-hydroxylation, and the appropriate combination of Val or Ala at position 114 with Ala or Ser at position 478 has recently been shown to convert P450 2B1 from an androstenedione and testosterone 16β-hydroxylase to a 15α-hydroxylase (Halpert, J. R., and He, Y.-A. (1993) J. Biol. Chem. 268, 4453-4457). All eight mutants containing the Val-363 → Ala substitution preferentially hydroxylated androstenedione and testosterone in the 15α-position and thus functionally resemble mouse P450 2A4. However, unlike P450 2A4, various single and multiple 2B1 mutants at positions 114, 363, and 478 mainly hydroxylated progesterone in the 16α- rather than 15α-position. By combining the Ile-114 → Ala substitution with a Phe-206 → Leu mutation (corresponding to Ala-117 and Leu-209 in P450 2A4), P450 2B1 was converted to a progesterone 15α-hydroxylase with retention of testosterone 15α-hydroxylase activity. These studies document the importance of residues 363 and 206 in determining the substrate specificity of P450 2B1 and strongly support the hypothesis that the judicious combination of a small number of discrete mutations can be used to confer new specificities on P450 enzymes.