Highly branched fatty acids, the main components of the preen-gland waxes of the domestic goose and the Muscovy duck, and their derivatives are promising chiral precursors for the synthesis of macrolide antibiotics. The key step in the utilisation of these compounds is their regioselective hydroxylation, which cannot be achieved in a classical chemical approach. Three P450 monooxygenases, CYP102A1, CYP102A2 and CYP102A3, demonstrating high turnover numbers in the hydroxylation of iso and anteiso fatty acids (>400 min(-1)), were tested for their activity towards these substrates. CYP102A1 from Bacillus megaterium and its A74G F87V L188Q triple mutant hydroxylate a variety of these substrates with high activity and regioselectivity. In all cases, the triple mutant showed much higher activities than the wild-type enzyme. The binding constants, determined for wild-type CYP102A1 and the triple mutant with tetramethylnonanol as substrate, were >200 microM and approximately 23 microM, respectively. Data derived from binding analysis support the differences in activity found for the wild-type CYP102A1 and the triple mutant. Surprisingly, CYP102A2 and CYP102A3 from Bacillus subtilis did not show any activity. Substrate binding spectra, recorded to investigate substrate accessibility to the enzyme's active sites, revealed that the substrates either could not access the active site of the Bacillus subtilis monooxygenases, or did not come into proximity with the heme.