Performing residue-swapping experiments between the highly conserved human steroidogenic proteins CYP11B1 and CYP11B2 we recently demonstrated that replacement of specific residues at position 301, 302 and 320 in the aldosterone-producing CYP11B2 protein for such residues that were specific for the highly similar cortisol-producing CYP11B1 protein elevated the 11beta-hydroxylase activity dramatically. Conversely, aldosterone synthesis in the triple mutant was severely impaired. Here we provide evidence that in a reciprocal experiment, CYP11B2-specific amino acids at position 320 and 335 endowed CYP11B1 with an 18-oxidase function amounting to 20% of the CYP11B2 wild-type activity, thus changing the specificity of steroid hydroxylation by only one point mutation. Combining substitutions at positions 296, 301, 302, 320, 335 and 339 did, however, not result in further enhancement. Paradoxically, 11beta-hydroxylation was not or only marginally affected in CYP11B1 mutants, indicating an alternative structural basis for this activity in CYP11B1 compared with the engineered CYP11B2 variant. Our results suggest that the sequence spanned by amino acids 301 and 335 constitutes part of the substrate-binding site in CYP11B1 and CYP11B2 as well. By constructing chimeric proteins we further investigated the effect of the C-terminal portions of both proteins and found that diverging residues at positions 471, 472, 492, 493 and 494 were insignificant for the stereospecificity and regiospecificity of steroid hydroxylation.