Four mutants of Thr-205 in cytochrome p450 2B1 were constructed and expressed in Escherichia coli. The Ser-, Ala-, and Val-mutants displayed stable reduced CO difference spectra and were able to metabolize 7-ethoxy-4-(trifluoromethyl)coumarin, testosterone, androstenedione, and benzphetamine. The Arg-mutant displayed an unstable reduced CO difference spectrum at 450 nm, was concomitantly converted to a denatured form with a peak at 422 nm, and showed no catalytic activity with any of the four substrates tested. The Ser-mutant displayed activity and metabolite profiles for testosterone and androstenedione similar to those of the wild-type p450 2B1 (WT). Substitution of Thr-205 with Ala or Val markedly suppressed the 16 beta-hydroxylation activity but exhibited little effect on the 16 alpha-hydroxylation activity for testosterone and androstenedione. Because 16 beta-hydroxylation activity of androgens is a specific p450 2B subfamily marker and residue 205 is located in the F helix, which forms the ceiling of the active site, we postulate that the gamma-hydroxyl side chain of Thr may play an important role in directing the 16 beta-face of testosterone and androstenedione toward the active site. Surprisingly, the Val-mutant retained full activity for benzphetamine demethylation. When mechanism-based inactivators for p450 2B1 were used to evaluate the susceptibility to inactivation, the Val-mutant was resistant to inactivation by 17 alpha-ethynylestradiol and less sensitive to inactivation by 2-ethynylnaphthalene compared with the WT enzyme. Our results demonstrate the importance of Thr-205 in determining substrate specificity and product formation as well as in influencing the susceptibility of p450 2B1 to mechanism-based inactivators.