Cannabinoids are part of an endogenous signaling system consisting of cannabinoid receptors and endogenous cannabinoids as well as the enzymatic machinery for their synthesis and degradation. Depolarization-induced suppression of excitation (DSE) is a form of cannabinoid CB(1) receptor-mediated inhibition of synaptic transmission that involves the production of the endogenous cannabinoid 2-arachidonoyl glycerol (2-AG). Both diacylglycerol lipase α (DAGLα) and DAGLβ can produce 2-AG in vitro, but evidence from knockout animals argues strongly for a predominant, even exclusive, role for DAGLα in regulation of 2-AG-mediated synaptic plasticity. What role, if any, might be played by DAGLβ remains largely unknown. Cultured autaptic hippocampal neurons exhibit robust DSE. With the ability to rapidly modulate expression of DAGLα and DAGLβ in these neurons with short hairpin RNA, they are well suited for a comparative study of the roles of each isoform in mediating DSE. We find that RNA interference knockdown of DAGLα substantially reduces autaptic DSE, shifting the "depolarization-response curve" from an ED(50) value of 1.7 seconds to 3.0 seconds. Surprisingly, DAGLβ knockdown diminishes DSE as much or more (ED(50) 6.4 seconds), suggesting that DAGLβ is also responsible for a portion of 2-AG production in autaptic neurons. Similarly, the two DAGLs both contribute to the production of 2-AG via group I metabotropic glutamate receptors. Our results provide the first explicit evidence for a role of DAGLβ in modulating neurotransmission.