The actions of different cholinergic agonists and antagonists were investigated on nociceptive afferents using the rat skin–saphenous nerve preparation,in vitro. Nicotine was able to weakly excite C-nociceptors and to induce a mild sensitization to heat stimulation (in 77% of tested fibers) in a dose-dependent manner (10⁻⁶to 10⁻⁵m), but it caused no alteration in mechanical responsiveness tested with von Frey hairs. Muscarine did not induce a significant nociceptor excitation, but almost all fibers exhibited a marked desensitization to mechanical and heat stimuli in a dose-dependent manner (from 10⁻⁶to 10⁻⁴m). The muscarinic effects could be prevented by the general muscarinic antagonist scopolamine (10⁻⁵m), by the M3 antagonist 1,1-dimethyl-4-diphenylacetoxypiperidium oxide (10⁻⁶m) co-applied with the M2 antagonist gallamine (10⁻⁵m), and by gallamine alone. As positive control we used the relatively M2-selective agonist arecaidine (10⁻⁶to 10⁻⁵m), obtaining a similar desensitizing effect as with muscarine. Finally, we performed an immunocytochemical study that demonstrated the presence of M2 but not M3 receptors in thin epidermal nerve fibers of the rat hairy skin. Altogether, these data demonstrate opposite effects of nicotinic and muscarinic receptor stimulation on cutaneous nociceptors. M2 receptor-mediated depression of nociceptive responsiveness may convey a therapeutic, i.e., analgesic or antinociceptive, potential.