The origin and three-dimensional distribution of substance P-immunoreactive structures in the rat stomach were investigated by means of the indirect immunofluorescence method using whole-mount tissue. The present study demonstrated that the myenteric plexus contained a very high concentration of substance P-immunoreactive fibers. It also contained cells that were moderately concentrated in the greater curvature, with those closer to the lesser curvature decreasing in number. In addition, a number of fibers ran parallel to the muscles in the circular and longitudinal muscle layers; these were distributed evenly throughout the entire stomach. We further found that most of the fibers located in the myenteric plexus originated from the greater splanchnic nerve from T5 to L2, but that some originated from intrinsic substance P-immunoreactive cells in the myenteric plexus. The fibers in the circular and longitudinal muscle layers originated from the cells in the myenteric plexus.