Our previous study showed that impairment of ascending and descending neural reflexes in the ileum of the c-kit mutant, W/W(V), mice is due to a loss of interstitial cells of Cajal present at the myenteric plexus region (ICC-MY) in the mutant. In the present study, cholinergic interneurons were thought to be involved in these pathways, since hexamethonium, an antagonist of the nicotinic ACh receptor, significantly inhibited both neural reflexes in wild type mice. Therefore, we examined whether the loss of ICC-MY affects cholinergic interneurons involved in these pathways. Immunohistochemistry with anti-choline acetyltransferase revealed that there was no difference in the numbers of immunopositive cells in the myenteric plexus region between the wild type and mutant mice. In addition, there was no difference in the extent of spontaneous and EFS-evoked ACh release from longitudinal muscle with myenteric plexus preparations between the wild type and mutant mice. Exogenously added nicotine induced contraction or relaxation of ileal circular muscle in the absence or presence of atropine, respectively, to a similar extent in both the wild type and mutant mice. These results suggest that loss of ICC-MY resulted in an impairment of the ascending and descending reflex pathways at the step before activation of cholinergic interneurons.