A cDNA encoding a naturally occurring variant of cytochrome P450 (P450) 2C3 that catalyzes the 6 beta- and 16 alpha-hydroxylation of progesterone exhibits six differences of nucleotide sequence leading to five amino acid substitutions from that encoding 2C3, a progesterone 16 alpha-hydroxylase that does not catalyze 6 beta-hydroxylation. Analysis of chimeric and mutant enzymes indicates that a Ser/Thr difference at position 364 underlies the difference between the two enzymes in 6 beta-hydroxylase activity as well as sensitivity to the inhibitor, 16 alpha-methylprogesterone. In addition, an Ile/Met difference at position 178 influences the apparent Km for progesterone. The two mutations, S364T and 1178M, together convert 2C3 to a form that exhibits kinetic properties which are similar to the 2C3v enzyme, and the reciprocal mutations in 2C3v convert it to an enzyme that resembles 2C3. Interestingly, position 364 of 2C3 maps to a substrate-contacting domain suggested by models for mammalian P450 enzymes based on the structure of P450cam. Ile178 is highly conserved among mammalian microsomal P450s with the exception of CYP4A and CYP19 enzymes which exhibit a Met at this alignment position.