1. Tramadol is a centrally acting analgesic with low opioid receptor affinity and therefore presumably other mechanisms of analgesic action. Tramadol inhibits noradrenaline uptake but since 5-hydroxytryptamine (5-HT) is also involved in the modulation of pain perception, we tested the effects of tramadol on 5-HT uptake and release in vitro. 2. Tramadol inhibited the uptake of [3H]-5-HT into purified rat frontal cortex synaptosomes with an IC50 of 3.1 microM. The (+)-enantiomer was about four times more potent than the (-)-enantiomer; the main metabolite of tramadol, O-desmethyltramadol, was about ten times less potent. 3. Rat frontal cortex slices were preincubated with [3H]-5-HT, then superfused and stimulated electrically. Tramadol facilitated the basal outflow of [3H]-5-HT, at concentrations greater than 1 microM, while the uptake inhibitor 5-nitroquipazine enhanced both basal and stimulation-evoked overflow. Effects of the (+)-enantiomer were more potent than either the racemate, the (-)-enantiomer or the principal metabolite. 4. The effects of tramadol on the basal outflow of [3H]-5-HT were almost completely abolished when the superfusion medium contained a high concentration of the selective 5-HT uptake blocker, 6-nitroquipazine. 5. The results provide evidence for an interaction of tramadol with the neuronal 5-HT transporter. An intact uptake system is necessary for the enhancement of extraneuronal 5-HT concentrations by tramadol indicating an intraneuronal site of action.