Abstract—Changes in the parameters of miniature endplate potential (MEPP) of mouse diaphragm caused by exogenous application of two classical endocannabinoids, 2-arachidonoylglycerol (2-AG) (1 μM) and anandamide (AEA) (30 μM), were compared. A slowly developing stable increase in the MEPP amplitude by 50%, without affecting the frequency of MEPPs, was caused by 2-AG. This effect was prevented by AM-251, an inverse agonist of CB1-receptors, as well as by vesamicol, a blocker of the vesicular acetylcholine (ACh) transporter. On the contrary, AEA did not cause significant changes in the MEPP amplitude but induced a slowly developing (within 2 h) increase in MEPP frequency by 75% on average. The effect of AEA was prevented by AM-251 (1 μM) as well as by blocking of L-type Ca2+-channels with nitrendipine (1 μM) and inhibition of PKA activity by H89 (1 μM). It was concluded that both 2-AG and AEA are able to exert a noncanonical facilitating presynaptic effect on spontaneous ACh release. Even though these endocannabinoids activate the same type of CB-receptors, their facilitating effects do not overlap and are strictly aimed either at potentiating the size of ACh quanta (in case of 2-AG) or increasing the frequency of MEPP (in case of AEA). We assume that different intracellular targets and signaling pathways may be involved in the differentiated facilitating effects of 2-AG and AEA in mouse neuromuscular junctions.