Tramadol is a well-known and effective analgesic. Recently it was shown that tramadol is also effective in human premature ejaculation. The inhibitory effect of tramadol on the ejaculation latency is probably due to its mechanism of action as a μ-opioid receptor agonist and noradrenaline/serotonin (5-HT) reuptake inhibitor. In order to test this speculation, we tested several doses of tramadol in a rat model of male sexual behavior and investigated two types of drugs interfering with the μ-opioid and the 5-HT system. First the μ-opioid receptor agonist properties of tramadol were tested with naloxone, a μ-opioid receptor antagonist. Second, the effects of WAY100,635, a 5-HT1A receptor antagonist, were tested on the behavioral effects of tramadol. Finally the effects of paroxetine, a selective serotonin reuptake inhibitor, combined with naloxone or WAY100,635 treatment, were compared to the effects of tramadol combined with these drugs. Results showed that naloxone, at a sexually inactive dose, could only partially antagonize the inhibitory effect of tramadol. Moreover, low and behaviorally inactive doses of WAY100,635, strongly decreased sexual behavior when combined with a behaviorally inactive dose of tramadol. Finally we showed that the effects of paroxetine on sexual behavior resembled the effects of tramadol, indicating that tramadol's inhibitory effects on sexual behavior are primarily and mainly caused by its SSRI properties and that its μ-opioid receptor agonistic activity only contributes marginally. These findings support the hypothesis that tramadol exerts inhibition of premature ejaculations in men by its 5-HT reuptake inhibiting properties.