The prokineticin Bv8, a small protein secreted by the skin of the Bombina variegata frog, is a potent agonist of both the identified prokineticin receptors, the G-protein-coupled PK-R1 and PK-R2. We found in this study that intraperiaqueductal grey (PAG) Bv8, 100 and 200 pmol per rat, exerted a pronociceptive action and caused opposite effects on the ongoing rostral ventromedial medulla (RVM) On- and Off-cell activities in rats. Bv8 increased and decreased the ongoing activity of RVM On and Off cells, respectively. Bv8 decreased tail flick latency and increased the pause and shortened the onset of the Off-cell pause. Bv8 did not change either the tail flick-induced On-cell burst of activity or the onset of On-cell peak firing. Microdialysis analysis, applied in combination with the plantar test, showed that intra-PAG perfusion with Bv8 (0.25 and 0.5 pm) increased GABA, but not glutamate, extracellular levels, and decreased thermoceptive thresholds. These findings show that stimulation of PAG PK-Rs might worsen pain perception and this effect is consistent with both RVM On- and Off-cell ongoing and tail flick-related activities, as well as with the increase induced by Bv8 in PAG GABA levels.