Intact endogenous cannabinoid signaling is involved in several aspects of drug addiction. Most importantly, endocannabinoids exert pronounced influence on primary rewarding effects of abused drugs, including exogenous cannabis itself, through the regulation of drug-induced increase in bursting activity of dopaminergic neurons in the ventral tegmental area (VTA). Previous electrophysiological studies have proposed that these dopaminergic neurons may release endocannabinoids in an activity-dependent manner to regulate their various synaptic inputs; however, the underlying molecular and anatomical substrates have so far been elusive. To facilitate understanding of the neurobiological mechanisms involving endocannabinoid signaling in drug addiction, we carried out detailed analysis of the molecular architecture of the endocannabinoid system in the VTA. In situ hybridization for sn-1-diacylglycerol lipase-alpha (DGL-alpha), the biosynthetic enzyme of the most abundant endocannabinoid, 2-arachidonoylglycerol (2-AG), revealed that DGL-alpha was expressed at moderate to high levels by most neurons of the VTA. Immunostaining for DGL-alpha resulted in a widespread punctate pattern at the light microscopic level, whereas high-resolution electron microscopic analysis demonstrated that this pattern is due to accumulation of the enzyme adjacent to postsynaptic specializations of several distinct morphological types of glutamatergic and GABAergic synapses. These axon terminal types carried presynaptic CB(1) cannabinoid receptors on the opposite side of DGL-alpha-containing synapses and double immunostaining confirmed that DGL-alpha is present on the plasma membrane of both tyrosine hydroxylase (TH)-positive (dopaminergic) and TH-negative dendrites. These findings indicate that retrograde synaptic signaling mediated by 2-AG via CB(1) may influence the drug-reward circuitry at multiple types of synapses in the VTA.