It has been previously shown that when expressed in the same cells the sole presence of CB1 receptors is able to potentiate OX1 receptor signaling to the ERK (extracellular signal- regulated kinase) pathway. This has been suggested to be due to heterodimerization of these receptors. On the other hand, we can here, in recombinant Chinese hamster ovary (CHO) cells, show that OX1 receptor activation leads to a strong release of 2-arachidonoylglycerol (2-AG), a potent endocannabinoid messenger, by diacylglycerol lipase action. The 2-AG released is able to activate CB1 receptors in paracrine manner. This is the first direct demonstration of 2-AG synthesis by orexin receptors, and it is in agreement with previous studies suggesting 2-AG as a retrograde synaptic transmitter in orexin responses in the central nervous system. We further set out to investigate the role of autocrine signaling behind the potentiation of ERK signaling in cells expressing both CB1 and OX1 receptors. OX1 signaling to ERK was strongly potentiated (elevated maximum response, left- shifted EC50-value) in OX1 and CB1 receptor-co- expressing cells. This potentiation was equally fully attenuated by blocking the CB1 receptors and by inhibiting diacylglycerol lipase, the enzyme responsible for 2-AG synthesis. Based on these data we suggest that the potentiation of ERK signaling in this expression system is due to autocrine action of 2-AG produced following OX1 activation rather than dimerization of CB1 and OX1.