We studied the role of calcium signaling in the subepithelial fibroblasts of rat small intestine which form cellular networks between the basal lamina of the epithelium and subepithelial capillaries. These cells are rich in actin bundles, and are coupled each other with gap junctions. Intracellular calcium level of the cultured subepithelial fibroblasts was transiently increased by the addition of endothelins, ATP, bradykinin, substance-P, serotonin etc, and their shape is changed from flat to stellate according to the increase of c-AMP.Moreover, intercellular calcium wave is propagated by the mechanical stimulation of a flat cell. Calcium wave propagation was not observed in the stellate cells. These characteristics are similar to those of cultured astrocytes. Calcium waves are believed to be transferred from cell to cell by gap junction-mediated diffusion of Ca^<2+> or IP3, or by extracellular components secreted from cells such as ATP/UTP via P2 purinergic receptors. In this study, we found that (1) the inhibitor of gap junctions did not inhibit calcium wave propagation. (2) Intracellular injection of IP3 did not induce calcium wave. (3) Significant difference was not observed in the number and size of gap junctions between flat and stellate cells by freeze-fracture and immunohistochemistry. (4) By the stretch stimulation, calcium wave propagation was occurred in the flat cells, but not in the stellate cells. These results indicate that calcium signaling in the subepithelial fibroblasts is probably mediated by release of ATP, not by gap junctions. ATP release from subepithelial fibroblasts may be related to the difference of cytoskeletal organization between flat cells and stellate cells.