Acetylcholine or carbachol stimulated cyclic GMP production in neuronal cultures from embryonic rat dorsal root ganglia but not in non-neuronal dorsal root ganglia cultures. Acetylcholine stimulation of cyclic GMP production was mediated by muscarinic receptors and required extracellular Ca2+. Basal cyclic GMP production and acetylcholine-evoked cyclic GMP production were attenuated by Methylene Blue, suggesting the involvement of soluble guanylate cyclase and nitric oxide synthase. L-NG-Monomethyl arginine attenuated basal, acetylcholine or carbachol-stimulated cyclic GMP production; this inhibition of acetylcholine and carbachol stimulation of cyclic GMP was reversed by L-arginine. These results suggest that a nitrosyl factor mediates basal, as well as acetylcholine- and carbachol-stimulated, cyclic GMP production. Selective destruction of small diameter neurons by capsaicin pretreatment of dorsal root ganglion neuronal cultures abolished acetylcholine and capsaicin stimulation of cyclic GMP, but did not affect sodium nitroprusside stimulation of cyclic GMP. These results suggest that acetylcholine evoked production of a nitrosyl factor in capsaicin-sensitive (small diameter) sensory neurons, which subsequently stimulated a soluble guanylate cyclase and cyclic GMP production in adjacent neuronal and/or non-neuronal cells. These results demonstrate that muscarinic agonists stimulate the nitric oxide-cyclic GMP signaling system in capsaicin-sensitive sensory neurons. Thus, the noxious character of acetylcholine when administered peripherally may be mediated by nitric oxide-cyclic GMP.