Transient increases in nucleus accumbens (NAc) dopamine concentration are observed when animals are presented with motivationally salient stimuli and are theorized to energize reward seeking. They arise from high-frequency firing of dopamine neurons in the ventral tegmental area (VTA), which also results in the release of endocannabinoids from dopamine cell bodies. In this context, endocannabinoids are thought to regulate reward seeking by modulating dopamine signaling, although a direct link has never been demonstrated. To test this, we pharmacologically manipulated endocannabinoid neurotransmission in the VTA while measuring transient changes in dopamine concentration in the NAc during reward seeking. Disrupting endocannabinoid signaling dramatically reduced, whereas augmenting levels of the endocannabinoid 2-arachidonoylglycerol (2AG) increased, cue-evoked dopamine concentrations and reward seeking. These data suggest that 2AG in the VTA regulates reward seeking by sculpting ethologically relevant patterns of dopamine release during reward-directed behavior. ► VTA endocannabinoid tone regulates neural mechanisms of cue-motivated reward seeking ► CB1 receptor antagonists decrease the neural mechanisms of reward seeking ► VTA levels of 2AG, not anandamide, facilitate the neural mechanisms of reward seeking ► Drugs targeting these systems might successfully be used in disorders of motivation To assess whether endocannabinoids regulate reward seeking, Oleson et al. manipulated endocannabinoid neurotransmission in the VTA and found alterations in cue-evoked dopamine levels in the NAc and reward seeking. These data suggest that endocannabinoid signaling in the VTA regulates reward seeking by sculpting dopamine release.