The mechanism by which CYP2B66 allele alters drug metabolism in vitro and in vivo is not fully understood. To test the hypothesis that altered substrate binding and/or catalytic properties contribute to its functional consequences, efavirenz 8-hydroxylation and bupropion 4-hydroxylation were determined in CYP2B6.1 and CYP2B6.6 proteins expressed without and with cytochrome b5 (Cyt b5) and in human liver microsomes (HLMs) obtained from liver tissues genotyped for the CYP2B66 allele. The susceptibility of the variant protein to inhibition was also tested in HLMs. Significantly higher V(max) and K(m) values for 8-hydroxyefavirenz formation and ∼2-fold lower intrinsic clearance (Cl(int)) were noted in expressed CYP2B6.6 protein (-b5) compared with that of CYP2B6.1 protein (-b5); this effect was abolished by Cyt b5. The V(max) and Cl(int) values for 4-hydroxybupropion formation were significantly higher in CYP2B6.6 than in CYP2B6.1 protein, with no difference in K(m), whereas coexpression with Cyt b5 reversed the genetic effect on these kinetic parameters. In HLMs, CYP2B66/6 genotype was associated with markedly lower V(max) (and moderate increase in K(m)) and thus lower Cl(int) values for efavirenz and bupropion metabolism, but no difference in catalytic properties was noted between CYP2B61/1 and CYP2B61/6 genotypes. Inhibition of efavirenz 8-hydroxylation by voriconazole was significantly greater in HLMs with the CYP2B66 allele (K(i) = 1.6 ± 0.8 μM) than HLMs with CYP2B61/1 genotype (K(i) = 3.0 ± 1.1 μM). In conclusion, our data suggest the CYP2B66 allele influences metabolic activity by altering substrate binding and catalytic activity in a substrate- and Cyt b5-dependent manner. It may also confer susceptibility to inhibition.