The effect of capsaicin-induced stimulation of afferent neurons on peristalsis and the possible neural mediators involved in this action were examined in the guinea-pig isolated ileum. The intraluminal pressure threshold for eliciting peristaltic waves was used to quantify facilitation (decrease in threshold) or inhibition (increase in threshold) of peristalsis. Capsaicin (0.1–1 μM) caused an initial short-lasting stimulation of peristalsis followed by a prolonged inhibition of peristaltic activity. Capsaicin (1 μM) was ineffective when the gut segments had been pretreated with 3.3 μM capsaicin, which is indicative of an afferent neuron-dependent action of the drug. In contrast, the abolition of peristalsis caused by a high concentration of capsaicin (33 μM) was fully reversible on removal and reproducible on readministration of capsaicin, a feature characteristic of a nonspecific depression of smooth muscle excitability. Baseline peristalsis and the excitatory/inhibitory effect of capsaicin (1 μM) on peristalsis remained unaltered by a combination of the tachykinin NK1 receptor antagonist ( + )-(2S, 3S)-3-(2-methoxybenzylamino)-2-phenyl piperidine (CP-99,994; 0.3 μM) and the tachykinin NK2 receptor antagonist L(-)-N-methyl-N[4-acetylamino-4-phenyl-piperidino-2-(3,4-dichlorophenyl)butyl]-benzamide (SR-48,968; 0.1 μM). Further experiments, performed in the presence of a low concentration of atropine (10 nM) showed that the catcitonin gene-related peptide (CGRP) antagonist human α-catcitonin gene-related peptide (8–37) [hCGRP (8–37); 10 μM] attenuated the delayed inhibitory effect of capsaicin on peristalsis, but did not influence baseline peristaltic activity and the capsaicin-induced facilitation of peristalsis. Blockade of nitric oxide (NO) synthesis by N G-nitro-l-arginine methylester (l-NAME, 300 μM) facilitated baseline peristaltic activity and reduced the delayed inhibition of peristalsis caused by capsaicin (1 μM) without affecting the initial peristalsis-stimulating action of capsaicin. The effects of l-NAME were prevented by l-arginine (1 mM). The data of the current study indicate that capsaicin-sensitive afferent neurons do not participate in the neural pathways subserving peristalsis in the guinea-pig small intestine, but modulate peristaltic activity upon stimulation with capsaicin. The initial stimulant action of capsaicin on peristalsis is independent of tachykinins acting via NK1 or NK2 receptors, while the delayed capsaicin-induced depression of peristalsis involves CGRP and NO.