Despite the emerging importance of human P450 2B6 in xenobiotic metabolism, thorough biochemical and biophysical characterization has been impeded as a result of low expression in Escherichia coli. Comparison with similar N-terminal truncated and C-terminal His-tagged constructs (rat P450 2B1dH, rabbit 2B4dH, and dog 2B11dH) revealed that P450 2B6dH showed the lowest thermal stability, catalytic tolerance to temperature, and chemical stability against guanidinium chloride-induced denaturation. Eleven P450 2B6dH mutants were rationally engineered based on sequence comparison with the three other P450 2B enzymes and the solvent accessibility of residues in the ligand-free crystal structure of P450 2B4dH. L198M, L264F, and L390P showed approximately 3-fold higher expression than P450 2B6dH. L264F alone showed enhanced stability against thermal and chemical denaturation compared with P450 2B6dH and was characterized further functionally. L264F showed similar preferential inhibition by pyridine over imidazole derivatives as P450 2B6dH. The Leu(264)-->Phe substitution did not alter the K(s) for inhibitors or the substrate benzphetamine, the K(m) for 7-ethoxy-4-(trifluoromethyl)coumarin, or the benzphetamine metabolite profiles. The enhanced stability and monodisperse nature of L264F made it suitable for isothermal titration calorimetry studies. Interaction of 1-benzylimidazole with L264F yielded a clear binding isotherm with a distinctly different thermodynamic signature from P450 2B4dH. The inhibitor docked differently in the binding pocket of a P450 2B6 homology model than in 2B4, highlighting the different chemistry of the active site of these two enzymes. Thus, L264F is a good candidate to further explore the unique structure-function relationships of P450 2B6 using X-ray crystallography and solution thermodynamics.