CYP3A4 and CYP3A5 exhibit significant overlap in substrate specificity, but can differ in catalytic activity and regioselectivity. To investigate their characteristics further, the enzymatic reactions of the two CYP3A enzymes were compared using midazolam, nifedipine, testosterone and terfenadine as substrates. Both CYP3A5 and CYP3A4 showed sigmoid and substrate inhibition patterns for testosterone 6beta-hydroxylation and terfenadine t-butylhydroxylation (TFDOH), respectively. In the other reactions, the kinetic model for CYP3A5 was not similar to that for CYP3A4. An inhibition study demonstrated that the interactions between alpha-naphthoflavone (alphaNF) and CYP3A substrates were different for the two CYP3A enzymes. alphaNF stimulated nifedipine oxidation catalysed by CYP3A5, but did not stimulate that catalysed by CYP3A4. alphaNF at less than 32 microM inhibited TFDOH catalysed by CYP3A5, but did not inhibit that catalysed by CYP3A4. These results indicate that CYP3A5 has different enzymatic characteristics from CYP3A4 in some CYP3A catalysed reactions.