The recreational drug 3,4-methylenedioxy-metamphetamine (MDMA; 'ecstasy') enhances serotonin and dopamine transmission. Repeated binge treatment with MDMA (5 mg/kg, 3 times daily, 3 h apart, once per week for 4 wk) was found to increase gene expression of S100B, a neurotrophic factor that modulates neuronal plasticity. Mutant mice overexpressing S100B were investigated to better understand how increased S100B expression may influence MDMA-induced biochemical and behavioural responses. In open-field behaviour, the later MDMA binges decreased rearing and thigmotaxis in S100B mutant mice compared to wild-type mice. In the elevated plus-maze, MDMA increased open-arm entries in both genotypes, but less tolerance to this effect was found in S100B mutant mice. Serotonin transporter (SERT) density was up-regulated in the substantia nigra in S100B mutant mice under baseline conditions. MDMA treatment increased SERT in wild-type mice, but did not further increase it in S100B mutant mice. Dopamine transporter density was down-regulated by MDMA in both genotypes in the striatum. 5-HT1B receptor density and G-protein coupling were higher in MDMA-treated S100B mutant mice than in saline-treated mutant mice and MDMA-treated wild-type mice in the medial globus pallidus. In conclusion, repeated MDMA treatment increases S100B mRNA. Certain explorative and anxiolytic-like behaviours in response to MDMA are potentiated and exhibit less tolerance in mice overexpressing S100B. The genotype-dependent behavioural responses are paralleled by adaptations in the serotonin system. Our data indicate that genetic differences in S100B gene expression may predispose individual differences in the responsivity to repeated intake of MDMA.