•  2‐Arachidonoylglycerol (2‐AG), one of the best‐characterized retrograde messengers at central synapses, has been thought to be produced ‘on demand’ through a diacylglycerol lipase α (DGLα)‐dependent pathway upon activation of postsynaptic neurons (on‐demand synthesis hypothesis). •  However, recent studies propose an alternative hypothesis that 2‐AG is pre‐synthesized by DGLα, stored in neurons, and released from such ‘pre‐formed pools’ without the participation of DGLα (pre‐formed pool hypothesis). •  To test these hypotheses, we examined the effects of acute pharmacological inhibition of DGL by a novel potent DGL inhibitor, OMDM‐188, on retrograde 2‐AG signalling. •  We found that 2‐AG‐mediated retrograde signalling was blocked after 1 h treatment with OMDM‐188 in acute slices from the hippocampus, striatum and cerebellum, and was blocked several minutes after OMDM‐188 application in cultured hippocampal neurons. •  These results fit well with the on‐demand synthesis hypothesis, rather than the pre‐formed pool hypothesis. Abstract  The endocannabinoid (eCB) 2‐arachidonoylglycerol (2‐AG) produced by diacylglycerol lipase α (DGLα) is one of the best‐characterized retrograde messengers at central synapses. It has been thought that 2‐AG is produced ‘on demand’ upon activation of postsynaptic neurons. However, recent studies propose that 2‐AG is pre‐synthesized by DGLα and stored in neurons, and that 2‐AG is released from such ‘pre‐formed pools’ without the participation of DGLα. To address whether the 2‐AG source for retrograde signalling is the on‐demand biosynthesis by DGLα or the mobilization from pre‐formed pools, we examined the effects of acute pharmacological inhibition of DGL by a novel potent DGL inhibitor, OMDM‐188, on retrograde eCB signalling triggered by Ca2+ elevation, Gq/11 protein‐coupled receptor activation or synergy of these two stimuli in postsynaptic neurons. We found that pretreatment for 1 h with OMDM‐188 effectively blocked depolarization‐induced suppression of inhibition (DSI), a purely Ca2+‐dependent form of eCB signalling, in slices from the hippocampus, striatum and cerebellum. We also found that at parallel fibre–Purkinje cell synapses in the cerebellum OMDM‐188 abolished synaptically induced retrograde eCB signalling, which is known to be caused by the synergy of postsynaptic Ca2+ elevation and group I metabotropic glutamate receptor (I‐mGluR) activation. Moreover, brief OMDM‐188 treatments for several minutes were sufficient to suppress both DSI and the I‐mGluR‐induced retrograde eCB signalling in cultured hippocampal neurons. These results are consistent with the hypothesis that 2‐AG for synaptic retrograde signalling is supplied as a result of on‐demand biosynthesis by DGLα rather than mobilization from presumptive pre‐formed pools.