1. Peristalsis is a co‐ordinated motor behaviour in which an anally propagated contraction of the circular muscle propels intraluminal contents. The role of excitatory motoneurons in peristalsis is well established; however the role of enteric inhibitory motoneurons is unknown. 2. A combination of a nitric oxide synthase inhibitor and apamin, which blocks relaxation of the circular muscle of guinea‐pig small intestine mediated by enteric inhibitory motoneurons, was used to investigate the role of inhibitory motoneurons in peristalsis in isolated segments of guinea‐pig small intestine. 3. N omega‐nitro‐L‐arginine methyl ester (L‐NAME, 400 microM) and N omega‐nitro‐L‐arginine (L‐NOArg, 100 microM) significantly reduced the threshold volume required to trigger emptying of the intestine. This effect was reversed by L‐arginine (4 mM) and L‐arginine alone increased the threshold volume for initiation of peristalsis. Sodium nitroprusside (0.1‐10 microM), which generates nitric oxide, also increased the threshold volume. L‐NAME, L‐NOArg, L‐arginine and sodium nitroprusside did not alter the maximal intraluminal pressure generated during emptying. Contraction of the longitudinal muscle during the initial phase of fluid infusion was significantly increased by L‐NAME and L‐NOArg and reduced by sodium nitroprusside (1 nM to 10 microM). 4. Apamin (0.5 microM) did not significantly alter the threshold volume necessary to initiate peristalsis or contraction of the longitudinal muscle. However, the maximal pressure generated when the intestine was emptying was significantly increased. Furthermore, short segments of circular muscle contracted apparently randomly, before peristaltic emptying was triggered. 5. A combination of L‐NAME and apamin completely disrupted peristalsis. Contractions of the circular muscle did not always start at the oral end. Stationary contractions as well as contractions propagating orally and anally were observed. 6. It is concluded that enteric inhibitory motoneurons are crucial for peristalsis to occur. They are important in setting the threshold at which peristaltic emptying is triggered, via nitric oxide. They are essential for the propagation of the circular muscle contraction, via an apamin‐sensitive mechanism of transmission. Contraction of the longitudinal muscle during peristalsis is partly inhibited by a nitric oxide‐mediated mechanism.