Tramadol is a central analgesic with low affinity for opioid receptors. A major active metabolite (O-desmethyl-tramadol) shows a higher affinity for opioid receptors than tramadol. The influence of naloxone and quinidine (a selective P450DB1 or CAP2D6 inhibitor) on tramadol effect was investigated crossover and double-blind vs placebo in healthy subjects. They received tramadol (100 mg p.o.), tramadol+naloxone (0.8 mg i.v.) and tramadol+quinidine (50 mg p.o.). Analgesia was assessed, after transcutaneous electrical stimulation, by a categorical numerical scale and by measurement of the antinociceptive effect at spinal level by R-III reflex. Analgesia peaked at 3 hours and lasted about 6 hours. The mean decrease in peak tramadol analgesia by naloxone was only 31%. Quinidine had no effect on the extent of tramadol analgesia, but inhibited tramadol induced myosis. We therefore conclude that tramadol analgesia is only partially mediated by a mu opioid agonist effect. Tramadol analgesia thus results from an action on opioid receptors other than the mu subtype and/or from nonopioid effects (monoaminergic system). Quinidine blockade of tramadol myosis suggests that the mu agonist component of tramadol effect results from its O-demethylation by the polymorphic P450DB1 enzyme.