Comparison of several recently determined X-ray crystal structures of mammalian cytochrome P450 family 2 enzymes suggests considerable movement of helix B_ when ligands bind. To investigate the functional role of helix B_ in P450 2B1, residues 100–109 were substituted with alanine and phenylalanine. Kinetic properties were examined with the typical 2B substrates 7-benzyloxyresoruWn, 7-ethoxy-4-triXuoromethylcoumarin, benzphetamine, and testosterone. Several mutants showed 2- to 3-fold changes in kcat values and signiWcant diVerences in catalytic eYciencies among the substrates examined, consistent with structural information suggesting that the helix B_ region can adopt multiple conformations with diVerent contact residues depending on the substrate. Homology modeling of P450 2B1 was performed based on an inhibitor-bound P450 2B4 structure, and the docking analyses were consistent with experimental results. The Wndings suggest that residues in the helix B_ region aVect regio- and stereoselective oxidation in P450 family 2 enzymes as well as substrate entry.