We evaluated the effects of intra-periaqueductal grey (PAG) N-arachidonoyl-serotonin (AA-5-HT), a compound with a "dual" ability to inhibit the fatty acid amide hydrolase (FAAH) and to antagonize transient receptor vanilloid type 1 (TRPV1) receptors, on endocannabinoid levels, rostral ventromedial medulla (RVM) ON and OFF cell activities, thermal nociception (tail flick in anaesthetized rats) and formalin-induced nocifensive responses in awake rats. AA-5-HT increased endocannabinoid levels in the PAG and induced analgesia. Paradoxically, it also depressed the RVM OFF cell, as well as the ON cell activities. The effect of AA-5-HT was mimicked by co-injecting the selective FAAH inhibitor URB597 and the selective TRPV1 antagonist I-RTX into the PAG, which also induced analgesia and inhibition of ON and OFF cell ongoing activities. The recruitment of "alternative" pathways, such as PAG-locus coeruleus (LC)-spinal cord might be responsible for AA-5-HT effect since we found evidence that (i) intra-PAG AA-5-HT increased LC neuron firing activities, and (ii) intrathecal phentolamine or ketanserin prevented the analgesic effect of AA-5-HT. Moreover, intra-PAG AA-5-HT prevented the changes in the ON and OFF cells firing activity induced by intra-paw formalin, and it inverted the formalin-induced increase in LC adrenergic cell activity. All AA-5-HT effects were antagonized by cannabinoid CB1 and TRPV1 receptor antagonists thus suggesting that co-localization of these receptors in the PAG can be an appropriate neural substrate for AA-5-HT-induced analgesia.