It is well known that the enteric nervous system plays a key role in the generation of gastrointestinal peristaltic movements. Recently, the networks of interstitial cells of Cajal (ICC) have been found to be essential in the generation of spontaneous gastrointestinal movements. However, the role of ICC in the mechanisms involved in the generation of peristaltic movements is still controversial. The aim of the present study was to reveal how pacemaker myenteric ICC (ICC-MY) and the enteric nervous system contribute to the mechanisms involved in the generation of intestinal peristalsis. We compared spontaneous peristaltic movements of the ileum in wild type (WT) mice with those in W/W^V mutant mice which are selectively deficient in ICC-MY. Simultaneous recordings were made from both the circular and longitudinal muscle of a 4-cm long segment of ileum under hydrostatic pressure of 0-0.5 cm H₂O. Mechanical activity and continuous video-images of the ileum were compared between WT and W/W^V mutant mice under control conditions, in the presence of N-nitro-L-arginine methyl ester (L-NAME) and after tetrodotoxin (TTX). In the WT mouse ileum, peristaltic waves to propagate from the oral to the anal end were frequently observed. The frequency of these peristaltic waves and their associated synchronous longitudinal and circular muscle contractions was increased by L-NAME. The peristaltic waves were abolished by TTX. In the W/W^V mutant mouse ileum, no peristaltic waves to propagate from the oral to the anal end were observed in control and even after L-NAME, although the local spontaneously generated longitudinal and circular muscle contractions were enhanced by L-NAME. These local contractions were not abolished by TTX. The results presented here suggested that ICC-MY are essential for the generation of spontaneous intestinal peristaltic movements. It is conceivable that ICC-MY may determine the polarity of the excitation of the intestine such that longitudinal and circular muscle contractions propagate from the oral to the anal end of the intestinal segments, although the question of why ICC-MY are necessary for the neural pathways remains unresolved.