The transient receptor potential vanilloid-1 (TRPV1) receptor is involved in peripheral and spinal nociceptive processing and is a therapeutic target for pain. We have shown previously that TRPV1 in the ventrolateral periaqueductal grey (VL-PAG) tonically contributes to brainstem descending antinociception by stimulating glutamate release into the rostral ventromedial medulla and OFF neuron activity. Since both opioid and vanilloid systems integrate and transduce pain sensation in these pathways, we investigated here the potential interaction between TRPV1 and µ-opioid receptors in the VL-PAG-RVM system. We found that the TRPV1 agonist, capsaicin, and the µ-receptor agonist DAMGO, when co-administered into the ventrolateral-PAG ad doses non-analgesic per se, produce: 1) antinociception in tests of thermal nociception; 2) stimulation of glutamate release into the RVM; 3) inhibition of ON neuron activity in the RVM. These effects were all antagonized by the TRPV1 and opioid receptor antagonists 5'-iodo-resiniferatoxin and naloxone, respectively, thus suggesting the existence of TRPV1-µ opioid interaction in the VL-PAG/RVM system. By using double immunofluorescence techniques, we found that TRPV1- and µ-opioid receptors are co-expressed in several neurons of the VL-PAG. These findings suggest that µ-receptors activation not only acts on inhibitory neurons to disinhibit PAG output neurons, but also interacts with TRPV1 activation at increasing glutamate release into the RVM, possibly by acting directly on PAG output neurons projecting to the RVM.