Repeated treatment with (+/-)3,4-methylenedioxymethamphetamine (MDMA) produces lasting depletions in serotonin (5-HT) markers in the brains of New and Old World monkeys. We have previously shown that macaques treated with MDMA (4 days, 10 mg/kg im, b.i.d.), exhibit an immediate, approximately 50% reduction of the 5-HT metabolite 5-hydroxyindoleacetic acid (5-HIAA) in cerebrospinal fluid (CSF) and 76-93% reductions in neocortical 5-HT content postmortem, but no lasting behavioral deficits under unchallenged conditions. Those monkeys were, however, more behaviorally sensitive to challenge with the 5-HT(2C) agonist 1-(3-chlorophenyl)piperazine (mCPP ) 1 year after the MDMA regimen. A rapid tryptophan-depletion protocol was employed to determine further if these MDMA-exposed monkeys are more behaviorally and electrophysiologically sensitive to perturbation of 5-HT neurotransmission. Acute intragastric administration of a tryptophan-deficient (TRYP(-)) mixture of amino acids resulted in significant reductions in CSF 5-HIAA in both MDMA-exposed and control monkeys. The TRYP(-) mixture also reduced the brainstem auditory-evoked potential (BSAEP) P4 latency in MDMA-exposed monkeys, similar to an effect observed for 13 weeks post-MDMA. Spatial working memory performance was improved by the TRYP(-) mixture in the control group, but not the MDMA-exposed monkeys. Other behavioral capabilities [visual recognition memory, reaction time (RT), reinforcer efficacy and fine motor control] were not significantly affected by the TRYP(-) mixture in either group of monkeys. Thus, underlying alterations in brain function resulting from prior exposure to MDMA, that were not observed under normal conditions, may be revealed following perturbation of 5-HT signaling. The BSAEP response and spatial working memory appear particularly sensitive to lasting functional differences associated with MDMA exposure.