The endogenous cannabinoid system has been shown to play a crucial role in controlling neuronal excitability and synaptic transmission. In this study we investigated the effects of a cannabinoid receptor (CB-R) agonist WIN 55,212-2 (WIN) on excitatory synaptic transmission in the rat ventral tegmental area (VTA). Whole-cell patch clamp recordings were performed from VTA dopamine (DA) neurons in an in vitro slice preparation. WIN reduced both NMDA and AMPA EPSCs, as well as miniature EPSCs (mEPSCs), and increased the paired-pulse ratio, indicating a presynaptic locus of its action. We also found that WIN-induced effects were dose-dependent and mimicked by the CB1-R agonist HU210. Furthermore, two CB1-R antagonists, AM281 and SR141716A, blocked WIN-induced effects, suggesting that WIN modulates excitatory synaptic transmission via activation of CB1-Rs. Our additional finding that both AM281 and SR141716A per se increased NMDA EPSCs suggests that endogenous cannabinoids, released from depolarized postsynaptic neurons, might act retrogradely on presynaptic CB1-Rs to suppress glutamate release. Hence, we report that a type of synaptic modulation, previously termed depolarization-induced suppression of excitation (DSE), is present also in the VTA as a calcium-dependent phenomenon, blocked by both AM281 and SR141716A, and occluded by WIN. Importantly, DSE was partially blocked by the D2DA antagonist eticlopride and enhanced by the D2DA agonist quinpirole without changing the presynaptic cannabinoid sensitivity. These results indicate that the two pathways work in a cooperative manner to release endocannabinoids in the VTA, where they play a role as retrograde messengers for DSE via CB1-Rs.