Peripheral inhibitory nerves are physiological regulators of the contractile behavior of visceral smooth muscles. One of the transmitters responsible for inhibitory neurotransmission has been reputed to be a purine, possibly ATP. However, the exact identity of this substance has never been verified. Here we show that beta-nicotinamide adenine dinucleotide (beta-NAD), an inhibitory neurotransmitter candidate, is released by stimulation of enteric nerves in gastrointestinal muscles, and the pharmacological profile of beta-NAD mimics the endogenous neurotransmitter better than ATP. Levels of beta-NAD in superfusates of muscles after nerve stimulation exceed ATP by at least 30-fold; unlike ATP, the release of beta-NAD depends on the frequency of nerve stimulation. beta-NAD is released from enteric neurons, and release was blocked by tetrodotoxin or omega-conotoxin GVIA. beta-NAD is an agonist for P2Y1 receptors, as demonstrated by receptor-mediated responses in HEK293 cells expressing P2Y1 receptors. Exogenous beta-NAD mimics the effects of the enteric inhibitory neurotransmitter. Responses to beta-NAD and inhibitory junction potentials are blocked by the P2Y1-selective antagonist, MRS2179, and the nonselective P2 receptor antagonists, pyridoxal phosphate 6-azophenyl-2',4'-disulfonic acid and suramin. Responses to ATP are not blocked by these P2Y receptor inhibitors. The expression of CD38 in gastrointestinal muscles, and specifically in interstitial cells of Cajal, provides a means of transmitter disposal after stimulation. beta-NAD meets the traditional criteria for a neurotransmitter that contributes to enteric inhibitory regulation of visceral smooth muscles.