Cytochrome P450 2B6 (CYP2B6) is primarily responsible for the metabolism of the anti-HIV drug efavirenz (EFV). We set out to explore the molecular basis for CYP2B6 activity toward EFV by examining the metabolism of eight EFV analogues. cDNA-expressed CYP2B6 formed monooxygenated metabolites from EFV analogues containing an intact oxazinone or oxazine ring, but not from analogues with a disrupted ring, suggesting this ring is important for metabolism of EFV by CYP2B6. Subsequent substrate depletion analysis of EFV and EFV analogues found to be CYP2B6 substrates revealed further differences between these CYP2B6 substrates. Compounds that were not found to be CYP2B6 substrates were still able to inhibit CYP2B6 activity toward a known substrate, bupropion, suggesting they do gain access to the CYP2B6 active site. Taken together, these data reveal structural characteristics of EFV, namely, the oxazinone ring, that are critical for CYP2B6 metabolism of compounds with the EFV chemical scaffold.