Cytochrome P450s IIA1 and IIA2, encoded by the CYP2A1 and CYP2A2 genes, display 88% amino acid sequence similarities. The dissimilarities of sequence between these two enzymes are primarily localized within four discrete regions of the polypeptides that are separated by regions of absolute sequence identity. IIA1 specifically hydroxylates the prototype substrate testosterone at the 7 alpha and 6 alpha position with a predominance of 7 alpha metabolite. IIA2, on the other hand, hydroxylates this steroid at eight positions on the molecule, with one of the most abundant metabolites being 15 alpha-hydroxytestosterone. To determine those amino acids responsible for the difference in testosterone hydroxylation specificities, chimeras were constructed between IIA1 and IIA2 cDNAs and expressed in cell culture using vaccinia-virus-mediated cDNA expression. Chimeras, in which the first 355 amino acids correspond to a single enzyme, maintain the specificity associated with that enzyme. Of six chimeras which have substitutions between amino acids 161 and 276, two are inactive and the remaining four give similar metabolite profiles, in which both 7 alpha and 15 alpha hydroxylation specificities have been lost. Two of these four chimeras are diametric apposites, suggesting that modification of either the N-terminal or central regions of the enzymes results in conformational changes that prevent the specific binding interactions responsible for the narrow regioselectivity associated with IIA1 and 15 alpha-hydroxytestosterone formation associated with IIA2.