The leucine 211 → phenylalanine (L211F) and leucine 211 → tyrosine (L211Y) mutant forms of cytochrome P450 3A4 have been generated by site-directed mutagenesis and expressed functionally in Escherichia coli. Substrate binding affinities (S50 values) for testosterone and 7-benzyloxy-4-trifluoromethylcoumarin (BFC) were similar for the mutants and wild-type CYP3A4 (49 and 21 μM for L211F, 35 and 20 μM for L211Y, and 33 and 20 μM for the wild type, respectively). For erythromycin, however, the Km values determined for the L211F and L211Y mutants were 2.4- and 10.5-fold higher than for the wild type. Furthermore, IC50 values for the inhibition of testosterone 6β-hydroxylation by erythromycin and troleandomycin for L211F were 2.4- and 3.7-fold higher, and those for L211Y were 3.4- and 9.2-fold higher than those measured for the wild type. Conversely, small inhibitors, such as diazepam, exhibited no significant difference in IC50 values between the wild type and the L211F and L211Y mutants. It is proposed that large substrates bound in the catalytic center of CYP3A4 with molecular volumes greater than ∼600 Å3 were less well accommodated in the altered active sites, resulting in lower association energies and increased IC50 values.