BACKGROUND: Methadone may cause respiratory depression and fatalities. Concomitant use of benzodiazepines in methadone-treated patients for chronic pain or as maintenance therapy for opiate abuse is common. However, the exact contribution of benzodiazepines to methadone-induced respiratory toxicity remains debatable. METHODS: We investigated the respiratory effects of the combination diazepam (20mg/kg)/methadone (5mg/kg) in the rat, focusing on methadone concentration/effect relationships. Respiratory effects were studied using arterial blood gases and whole-body plethysmography. Plasma concentrations of both R- and S-methadone enantiomers were measured using high-performance liquid chiral chromatography coupled to mass spectrometry. To clarify mechanisms of diazepam/methadone interaction, methadone metabolism was investigated in vitro using rat liver microsomes. RESULTS: Diazepam/methadone co-administration significantly increased methadone-related effects on inspiratory time (p<0.001) but did not significantly alter the other respiratory parameters when compared with methadone alone, despite significant increase in the area under the curve of plasma R-methadone concentrations measured during 240min (p<0.05). Diazepam/methadone co-incubation with microsomes in vitro resulted in a significant inhibition of methadone metabolism (p<0.01), with 50%-inhibitory diazepam concentrations of 25.02±0.18μmol/L and 25.18±0.23μmol/L for R- and S-methadone, respectively. CONCLUSION: We concluded that co-administration of high-doses of diazepam and methadone in rats is not responsible for additional respiratory depression in comparison to methadone alone, despite significant metabolic interaction between the drugs. In humans, although our experimental data may suggest the relative safety of benzodiazepine/methadone co-prescription, physicians should remain cautious as other underlying conditions may enhance this drug-drug interaction.