A Ser/Thr difference at position 364 underlies a phenotypic difference between naturally occurring variants of microsomal cytochrome P450 2C3 in their capacity to catalyze the 6 beta-hydroxylation of progesterone, as well as in their sensitivity to the inhibitor 16 alpha-methyl-progesterone. Position 364 of P450 2C3 maps to a substrate contacting domain suggested by models for mammalian P450 enzymes based on the structure of P450,101. In this study, Thr-364 of P450 2C3v, a progesterone 6 beta- and 16 alpha-hydroxylase, was replaced by Gly, Asp, Asn, Val, Leu, or Ile. The latter three amino acids did not alter the regiospecificity of P450 2C3v, whereas the Gly, Asp, and Asn substitutions each produced enzymes with properties that correspond closely to the Ser mutant that catalyzes 16 alpha-hydroxylation but not 6 beta-hydroxylation. The former are distinguished from the latter amino acids by their greater hydrophobicity and size. In contrast, the 16 alpha-hydroxylase activity could be greatly diminished by the introduction of an alanine replacement for Val-113. This mutation conferred progesterone 21- and 17 alpha-hydroxylase activity to P450 2C3v at the expense of 16 alpha-hydroxylase activity, leaving the 6 beta-hydroxylase activity largely unaffected. Compound mutants displayed the additive effects of the two mutations. These results are consistent with two distinct orientations for the binding of progesterone to P450 2C3v, resulting in 6 beta- and 16 alpha-hydroxylation, respectively. The binding of progesterone in these two orientations can be modulated relatively independently by modifications of the two key amino acid residues at 113 and 364.