Adolescents and young adults disproportionately abuse 3,4-methylenedioxymethamphetamine (MDMA; ‘Ecstasy’); however, since most MDMA research has concentrated on adults, the effects of MDMA on the developing brain remain obscure. Therefore, we evaluated place conditioning to MDMA (or saline) during late adolescence and assessed anxiety-like behavior and monoamine levels during abstinence. Rats were conditioned to associate 5 or 10 mg/kg MDMA or saline with contextual cues over 4 twice-daily sessions. Five days after conditioning, anxiety-like behavior was examined with the open field test and brain tissue was collected to assess serotonin (5-hydroxytryptamine, 5-HT) and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) in the dorsal raphe, amygdala, and hippocampus by high-pressure liquid chromatography (HPLC). In a separate group of rats, anxiety-like and avoidant behaviors were measured using the light–dark box test under similar experimental conditions. MDMA conditioning caused a place aversion at 10, but not at 5, mg/kg, as well as increased anxiety-like behavior in the open field and avoidant behavior in light–dark box test at the same dose. Additionally, 10 mg/kg MDMA decreased 5-HT in the dorsal raphe, increased 5-HT and 5-HIAA in the amygdala, and did not alter levels in the hippocampus. Overall, we show that repeated high (10 mg/kg), but not low (5 mg/kg), dose MDMA during late adolescence in rats increases anxiety-like and avoidant behaviors, accompanied by region-specific alterations in 5-HT levels during abstinence. These results suggest that MDMA causes a region-specific dysregulation of the serotonin system during adolescence that may contribute to maladaptive behavior.