OBJECTIVE: The authors’ goal was to determine if the reported clinical adverse interaction of flunitrazepam and buprenorphine was caused by inhibition of drug metabolism.METHOD: Inhibition of flunitrazepam metabolism by buprenorphine and norbuprenorphine were determined in three human liver microsome preparations carrying the CYP2C19*1/*1 allele. Omeprazole metabolism mediated by CYP2C19 and CYP3A4 was used as a control reaction. Apparent Ki values were determined.RESULTS: Norbuprenorphine did not inhibit the metabolism of flunitrazepam or omeprazole. Buprenorphine inhibited the formation of CYP3A4-mediated pathways of 3-hydroxyflunitrazepam and omeprazole sulfone formation (Ki 118 and 16 μM) in human liver microsomes. Corresponding values were 38 and 90 μM in cDNA-expressed CYP3A4 microsomes. Projected in vivo inhibition of CYP3A4-mediated metabolism of flunitrazepam by buprenorphine is 0.1%–2.5%. Estimated inhibition of buprenorphine N-dealkylation by flunitrazepam in vivo is 0.08%.CONCLUSIONS: The clinical interaction of flunitrazepam and buprenorphine is likely based on a pharmacodynamic mechanism.