Wild-type cytochrome P450 monooxygenase from Bacillus megaterium (P450 BM-3) has a low hydroxylation activity for beta-ionone (< 1 min(-1)). Substitution of phenylalanine by valine at position 87 led to a more than 100-fold increase in beta-ionone hydroxylation activity (115 min(-1)). Enzyme activity could be further increased by both site-directed and random mutagenesis. The mutant R47L Y51F F87V, designed by site-directed mutagenesis, and the mutant A74E F87V P386S, obtained after two rounds of error-prone polymerase chain reaction, exhibited an increase in activity of up to 300-fold compared to the wild-type enzyme. The triple mutant R47 LY51F F87V exhibited moderate enantioselectivity, forming (R)-4-hydroxy-beta-ionone with an optical purity of 39%. All mutants regioselectively converted beta-ionone into 4-hydroxy-beta-ionone. The regioselectivity is determined amongst others by the absolute configuration of the substrate.