Recombinant P450 2B1 wild-type and the active-site mutants I114V, F206L, V363A, V363L, and G478S were purified and studied. The efficiency of coupling of reducing equivalents to 7-hydroxycoumarin formation was decreased for all the mutants except I114V. Uncoupling to H2O was increased for F206L, V363A, and G478S, decreased for V363L, and unchanged for I114V. Uncoupling to H2O2 was increased for V363L and decreased for I114V, F206L, and V363A. The findings from this study provide firm biochemical evidence that residues 206, 363, and 478 comprise part of the substrate binding site of P450 2B1.