The ventral tegmental area (VTA) is the origination zone for dopaminergic neurons involved in reward and addictive properties of a variety of abused substances. A major excitatory projection to VTA neurons originates in the medial prefrontal cortex, and several lines of evidence suggest that glutamatergic synapses on VTA neurons are activated and modified during exposure to psychostimulant drugs. Here, we report for the first time that amphetamine depresses excitatory glutamatergic synaptic transmission onto VTA neurons in the midbrain slice preparation. Unexpectedly, this depression is mediated not by activation of dopamine receptors, but instead by activation of serotonin receptors. Our findings suggest that an acute effect of amphetamine exposure is the release of serotonin in the VTA, which in turn modulates excitation of VTA neurons. This process may be an important early component of permanent changes occurring in the reward pathway that contribute to drug addiction.