Intercellular communication within the musculature of the canine colon was studied by examining the results of neurobiotin diffusion after injection of the tracer into smooth muscle cells at different locations within the muscle layer. Circular muscle at the submucosal surface, circular muscle adjacent to the myenteric plexus, and longitudinal muscle demonstrated different degrees of time-dependent tracer spread. At the submucosal surface, tracer spread was rapid, extensive, and unimpeded by connective tissue septa. At the myenteric side, tracer spread was also extensive but was much slower and confined to bundles of cells bordered by septa. In contrast to previous studies that suggest an absence of gap junctions at the myenteric side of the circular muscle, the neurobiotin spread indicates full metabolic coupling of all circular smooth muscle cells. Furthermore, in contrast to the belief that longitudinal muscle is completely devoid of gap junctions, tracer spread occurred between cells in this layer, although neurobiotin diffusion was very limited, nonuniform, and slow. In each area of the musculature studied, tracer spread was inhibited by octanol. When very long injection and wait times were implemented at the submucosal surface of the circular muscle, neurobiotin was observed to cross septa through the network of interstitial cells of Cajal, indicating that it is this network that provides communication between lamellae.