The molecular basis for reversible inhibition of rabbit CYP2B4 and CYP2B5 and rat CYP2B1 by phenylimidazoles was assessed with active-site mutants and new three-dimensional models based on the crystal structure of CYP2C5. 4-Phenylimidazole was 17- to 32-fold more potent toward CYP2B4 and CYP2B1 than CYP2B5. The 3D models, along with site-directed mutagenesis data, revealed the importance of residue 114 for sensitivity to inhibition of all three CYP2B enzymes. Besides Ile 114, Val 367 was also found to be critical for inhibition of CYP2B4 and CYP2B1. The most interesting new insights were obtained from analysis of the CYP2B5 model and the CYP2B5 active-site mutants. Simultaneous substitution of residues 114, 294, 363, and 367 with the corresponding residues of CYP2B4 decreased the IC(50) value for inhibition by 4-phenylimidazole 12-fold. Docking 4-phenylimidazole into the models of CYP2B5 mutants demonstrated that the inhibitor-binding site is strongly influenced by residue-residue interactions, especially between residues 114 and 294. A chlorine substitution at position 4 of the phenyl moiety of 4- and 1-phenylimidazole resulted in IC(50) values 95- and 130-fold lower for CYP2B4 than for CYP2B5, respectively, suggesting that these compounds are selective inhibitors of CYP2B4. Overall, the study revealed that differences in the determinants of inhibition between CYP2B4 and CYP2B5 are caused not only by single residue inhibitor contacts but also by residue-residue interactions. This new generation of CYP2B models may provide valuable information for the design of selective inhibitors of human CYP2B6 and for the development of drugs that avoid drug interactions due to P450 inhibition.