In gastrointestinal muscles special cells, referred to an interstitial cells, may be involved in pacemaking and transduction of inputs from the enteric nervous system. We have used a modification of the NADH diaphorase method to characterize the distribution of interstitial cells in the muscularis externa of the canine colon. The staining product of the NADH diaphorase reaction is useful because it allows light and electron microscopic studies to be performed with the same marker. Therefore rigorous identification of the cells observed at the light microscopic level could be made by electron microscopy. We were able to label at least three classes of interstitial cells: (1) at the submucosal surface of the circular muscle layer; (2) within the thickness of the circular and longitudinal muscle layers; and (3) in the region of the myenteric plexus. This technique also labeled cell bodies and initial segments of processes of Dogiel type II neurones in enteric ganglia. Nerve fibres within the muscle layers did not exhibit NADH diaphorase activity. This study has identified the interstitial cells within the circular and longitudinal muscle layers and shows the arrangement of these cells in a three-dimensional network.