By replacing specific amino acids at positions 112, 147 and 152 of the human aldosterone synthase (CYP11B2) with the corresponding residues from human, mouse or rat 11beta-hydroxylase (CYP11B1), we have been able to investigate whether these residues belong to structural determinants of individual enzymatic activities. When incubated with 11-deoxycorticosterone (DOC), the 11beta-hydroxylation activity of the mutants was most effectively increased by combining D147E and I112P (sixfold increase). The two substitutions displayed an additive effect. The same tendency can be observed when using 11-deoxycortisol as a substrate, although the effect is less pronounced. The second step of the CYP11B2-dependent DOC conversion, the 18-hydroxylation activity, was not as strongly increased as the 11beta-hydroxylation potential. Activity was unaffected by D147E, whereas the single mutant I112P displayed the most pronounced activation (70% enhancement), thus causing different increasing effects on the first two enzymatic reaction steps. A slightly enhanced aldosterone synthesis from DOC could be measured due to increased levels of the intermediates. However, the 18-oxidation activity of all the mutants, except for I112S and D147E, was slightly reduced. The strongly enhanced 18-hydroxycorticosterone and aldosterone formation observed in the mutants provides important information on a possible role of such amino-acid replacements in the development of essential hypertension. Furthermore, the results indicate the possibility of a differential as well as independent modification of CYP11B2 reaction steps. The combination of functional data and computer modelling of CYP11B2 suggests an indirect involvement of residue 147 in the regulation of CYP11B isoform specific substrate conversion due to its location on the protein surface. In addition, the results indicate the functional significance of amino-acid 112 in the putative substrate access channel of human CYP11B2. Thus, we present the first example of substrate recognition and conversion being attributed to the N-terminal part of human CYP11B2.