The effects of capsaicin on [³H]acetylcholine release and muscle contraction were studied on the myenteric plexus-longitudinal muscle preparation of the guinea-pig ileum preincubated with [³H]choline. Capsaicin concentration-dependently increased both basal [³H]acetylcholine release (pEC₅₀ 7.0) and muscle tone (pEC₅₀ 6.1). The facilitatory effects of capsaicin were antagonized by 1 μM capsazepine (pK _B 7.0 and 7.6), and by the combined blockade of NK₁ and NK₃ tachykinin receptors with the antagonists CP99994 plus SR142801 (each 0.1 μM). This suggests that stimulation by capsaicin of TRPV1 receptors on primary afferent fibres causes a release of tachykinins which, in turn, mediate via NK₁ and NK₃ receptors an increase in acetylcholine release. The capsaicin-induced acetylcholine release was significantly enhanced by the NO synthase inhibitor L-N^G-nitroarginine (100 μM). This indicates that tachykinins released from sensory neurons also stimulate nitrergic neurons and thus lead, via NO release, to inhibition of acetylcholine release. Capsaicin concentration-dependently reduced the electrically-evoked [³H]acetylcholine release (pEC₅₀ 6.4) and twitch contractions (pEC₅₀ 5.9). The inhibitory effects were not affected by either capsazepine, NK₁ and NK₃ receptor antagonists, the cannabinoid CB₁ antagonist SR141716A or by L-N^G-nitroarginine. Desensitization of TRPV1 receptors by a short exposure to 3 μM capsaicin abolished the facilitatory responses to a subsequent administration, but did not modify the inhibitory effects. In summary, capsaicin has a dual effect on cholinergic neurotransmission. The facilitatory effect is indirect and involves tachykinin release and excitation of NK₁ and NK₃ receptors on cholinergic neurons. The inhibition of acetylcholine release may be due to a decrease of Ca²⁺ influx into cholinergic neurons.