Cytochrome P450 (CYP) 3A4 metabolizes aflatoxin B1 (AFB1) to AFB1-exo-8,9-epoxide (8,9-epoxidation) and aflatoxin Q1 (AFQ1; 3$alpha$-hydroxylation) simultaneously. We investigated whether each metabolite was formed via its own binding site of CAP3A4 active site. Kinetics of the formation of the two metabolites were sigmoidal and consistent with the kinetics of substrate activation. The HIll model predicted that two substrate binding wites are involved in the oxidationof AFB1 by CYP3A4. Dehydronifedipine, a metabolite of nifedipine generated by CYP3A4, inhibited the formation of AFQ1 without any inhibition in the formation of AFB1-exo-8,9-epoxidation. Dehydronifedipine was found to act as a reversible competitive inhibitor against 3$alpha$-hydroxylation of AFB1. Vmax and S0.5 of the 8,9-epoxidation were not changed in the presence of 0, 50, or 100 $mu extrm{M}$ dehydronifedipine. S0.5 of 3$alpha$-hydroxylation was increased from 58$pm$4 $mu extrm{M}$ to 111$pm$8 $mu extrm{M}$ in the presence of 100 $mu extrm{M}$ nifedipine whereas Vmax was not changed. These results suggest that there exist two independent binding sites in the active site of CAP3A4 . One binding site is responsible for AFB1-exo-8,9-epoxidation and the other is involved in 3$alpha$-hydroxylation of AFB1. Dehydronifedipine might selectively bind to the site which is responsible for the formation of AFQ1 in the active site of CYP3A4.