Rationale: 3,4-Methylenedioxymethamphetamine (MDMA) produces a long-term depletion of serotonin (5-HT) in the rat brain; this depletion may have some functional consequences. Objective: The aim of the present study was to evaluate the acute effects of MDMA on the extracellular concentrations of dopamine and 5-HT, body temperature and the 5-HT behavioral syndrome in rats 7 days following a neurotoxic regimen of MDMA. Methods: One week after the rats were treated with a neurotoxic regimen of MDMA (10 mg/kg, IP, every 2 h for a total of four injections), the rats were injected with a subsequent injection of MDMA. In vivo microdialysis combined with HPLC was utilized to measure the extracellular concentration of 5-HT and dopamine in the striatum. The increase in body temperature was determined by rectal temperature measurements, and the 5-HT behavioral syndrome was scored using a rating scale following the administration of MDMA. Results: The neurotoxic regimen produced a 45% reduction in brain 5-HT concentrations. The magnitude of the MDMA-induced increase in the extracellular concentration of 5-HT, but not dopamine, in the striatum produced by an acute injection of MDMA (7.5 mg/kg, IP) was reduced in rats treated previously with the neurotoxic regimen of MDMA when compared with that in control animals. In addition, the magnitude of the 5-HT behavioral syndrome, as well as the hyperthermic response, produced by MDMA was markedly diminished in rats that had previously received the neurotoxic regimen of MDMA. Conclusions: It is concluded that the long-term depletion of brain 5-HT produced by MDMA is accompanied by impairments in 5-HT function, as evidenced by the deficits in the neurochemical, thermal and behavioral responses to subsequent MDMA administration.