Anatomical relationships between c-Kit-positive cells and nitric oxide synthase-positive nerves in the small intestine were examined by double-labeling immunohistochemistry. Cryosections and whole mount preparations of the guinea-pig small intestine were double-immunolabeled using anti-c-Kit and neuronal nitric oxide synthase antibodies, and were observed using confocal laser scanning microscopy. The c-Kit-like immunoreactivity constituted dense reticular networks in the deep muscular plexus and myenteric plexus of the intestinal wall. The nitric oxide synthase-like immunoreactivity occurred in the circular muscle layer, most densely at the deep muscular plexus, as well as within the ganglion strands or connecting strands of the myenteric plexus. Close association between c-Kit-like immunoreactivity and nitric oxide synthase-like immunoreactivity was evident in the deep muscular plexus. Specimens immunolabeled with the anti-nitric oxide synthase antibody were further examined under transmission electron microscopy. Axon profiles with nitric oxide synthase-like immunoreactivity lay closely adjacent to the interstitial cells in the deep muscular plexus as well as to smooth muscle cells of the circular muscle layer, whereas there was a considerable distance (> 500 nm) between interstitial cells and axon profiles with nitric oxide synthase-like immunoreactivity in the myenteric plexus. These results suggest that the interstitial cells in the deep muscular plexus serve as mediators of the nitrergic neurotransmission to the musculature in the small intestine, playing a role in the regulation of intestinal movement.