3,4-Methylenedioxymethamphetamine (MDMA) use and abuse have been increasing worldwide. Of concern, exposure to high doses of MDMA decreases several markers of serotonin (5HT) neurotransmission and produces deficits in tissue levels of 5HT. Studies in laboratory animals have been conducted primarily following large doses (20.0–80.0 mg/kg) of experimenter-administered MDMA, but it is unclear whether similar persistent deficits in tissue 5HT levels are produced following self-administration. In this study, tissue levels of 5HT in the frontal cortex, striatum and hippocampus were measured following different levels of self-administered MDMA. For both groups, responding was initially reinforced by an infusion of 1.0 mg/kg/infusion MDMA. The dose was reduced to 0.5 mg/kg/infusion once 90 infusions had been self-administered. For the two groups, testing continued until either a total of 165 or 315 mg/kg had been self-administered. Assays were conducted either 2 or 10 weeks following the last self-administration session. The lower dose exposure regimen failed to significantly decrease 5HT levels in any brain region. The higher dose exposure, however, decreased 5HT levels by 30–35% in all three brain regions 2 weeks, but not 10 weeks, following self-administration. Thus, MDMA self-administration produced dose- and time-dependent deficits in tissue levels of 5HT, suggesting that similar deficits would be produced in humans who use and abuse the drug.