On-demand postsynaptic synthesis and release of endocannabinoid lipids and subsequent binding to presynaptic CB1 receptors (CB1Rs) mediates short and long-term depression (LTD) of excitatory transmission in many brain regions. However, mechanisms involved in the synthesis of the endocannabinoid 2-arachidonoylglycerol (2-AG) by diacylglycerol lipase α (DGLα) are poorly understood. Since Gq-coupled receptor activation can stimulate production of a major DGL substrate 1-stearoyl-2-arachidonoyl-sn-glycerol (SAG) by PLCβ, we sought to determine if 2-AG biosynthesis was limited only by a lack of substrate availability, or if other pathways, such as Ca(2+) signaling, also need to be simultaneously engaged. To address this question, we loaded medium spiny neurons of the dorsolateral striatum with SAG while monitoring excitatory synaptic inputs. SAG-loading had no significant effect on evoked excitatory synaptic currents when cells were voltage-clamped at -80 mV. However, depolarization of MSNs to -50 mV revealed a SAG-loading dependent decrease in the amplitude of excitatory currents that was accompanied by an increase in paired pulse ratio, consistent with decreased glutamate release. Both effects of loading SAG at -50 mV were blocked by chelation of postsynaptic Ca(2+) using BAPTA or by bath application of tetrahydrolipstatin (THL), a DGL inhibitor. Loading of SAG into glutamatergic pyramidal neurons of the amygdala similarly inhibited excitatory synaptic inputs and increased the PPR. SAG-induced depression was absent in both regions from mice lacking CB1Rs. These data show that increasing substrate availability alone is insufficient to drive 2-AG mobilization and that DGL-dependent synaptic depression via CB1R activation requires postsynaptic Ca(2+) signals.