While several studies have investigated the effect of proton pump inhibitors (PPI) on the activities of specific cytochrome P450s (CYP450), most studies used model substrates. Here we investigate the effect of PPIs on the N‐dealkylation of buprenorphine, a CYP3A4 and 2C8 driven reaction. An initial screen was performed using three concentrations of PPI in human liver microsomes (HLM) with or without a 15‐minute pre‐incubation of the HLM with inhibitor and a source of NADPH. For five PPIs, the following apparent IC50s (µM) were determined without (‐) or with (+) 15 minute pre‐incubation: omeprazole, (‐) 20.9, (+) 2.5; esomeprazole, (‐) 9.8, (+) < 1.0; lansoprazole, (‐) > 100, (+) > 100; pantoprazole, (‐) 120, (+) 16.2; and rabeprazole (‐) 79.4, (+) 60.3. For all but lansoprazole, pre‐incubation effects suggest time‐dependent inhibition (TDI). Subsequent studies with cDNA‐expressed CYPs (rCYP) were conducted with 6 or more concentrations of inhibitor to determine an IC50. Respective IC50s (µM) determined with rCYP3A4 and 2C8 were: omeprazole, 220 and 65; esomeprazole, 90 and 65; lansoprazole, > 100 and 90; pantoprazole, > 200 and 200; and rabeprazole, 150 and 70. TDI tests were also conducted for specific PPIs with a dual incubation system. From a primary incubation with 3 or more inhibitor concentrations, aliquots then transferred (and diluted) after varying pre‐incubation times to a secondary system with buprenorphine and additional NADPH. These were conducted for omeprazole, esomeprazole, lansoprazole, pantoprazole and rabeprazole with rCYP3A4, and omeprazole and esomeprazole with rCYP2C8. Only rabeprazole produced pre‐incubation time‐ and inhibitor concentration‐dependent decreases in CYP3A4 activity (Ki = 2.6 µM, kinact = 0.052 min‐1). None of the others demonstrated TDI, thus an enigma arose between HLM and rCYP TDI results. When we performed the dual incubation using esomeprazole and HLM, inhibition was pre‐incubation time‐ and inhibitor concentration‐dependent (Ki = 20.9 µM and kinact = 0.0265 min‐1). Therefore, we hypothesize that metabolism of esomeprazole (and likely omeprazole and pantoprazole) by another CYP produces a potent inhibitor for CYP3A4 and/or CYP2C8 metabolism of buprenorphine.Grant Funding Source: Supported by National Institute of Justice Award #: 2011‐DN‐BX‐K532