Nucleotides contribute to the sensation of acute and chronic pain, but it remained enigmatic which G protein-coupled nucleotide (P2Y) receptors and associated signaling cascades are involved. To resolve this issue, nucleotides were applied to dorsal root ganglion neurons under current- and voltage-clamp. Adenosine triphosphate (ATP), adenosine diphosphate (ADP), and uridine triphosphate (UTP), but not uridine diphosphate (UDP), depolarized the neurons and enhanced action potential firing in response to current injections. The P2Y 2 receptor preferring agonist 2-thio-UTP was equipotent to UTP in eliciting these effects. The selective P2Y 1 receptor antagonist MRS2179 largely attenuated the excitatory effects of ADP, but left those of 2-thio-UTP unaltered. Thus, the excitatory effects of the nucleotides were mediated by 2 different P2Y receptors, P2Y 1 and P2Y 2. Activation of each of these 2 receptors by either ADP or 2-thio-UTP inhibited currents through K V7 channels, on one hand, and facilitated currents through TRPV 1 channels, on the other hand. Both effects were abolished by inhibitors of phospholipase C or Ca 2+-ATPase and by chelation of intracellular Ca 2+. The facilitation of TRPV 1, but not the inhibition K V7 channels, was prevented by a protein kinase C inhibitor. Simultaneous blockage of K V7 channels and of TRPV 1 channels prevented nucleotide-induced membrane depolarization and action potential firing. Thus, P2Y 1 and P2Y 2 receptors mediate an excitation of dorsal root ganglion neurons by nucleotides through the inhibition of K V7 channels and the facilitation of TRPV 1 channels via a common bifurcated signaling pathway relying on an increase in intracellular Ca 2+ and an activation of protein kinase C, respectively. © 2011 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.