It was reported that a signaling pathway of platelet-derived growth factors (PDGFs) and their receptors (PDGFRs) played a critical role in the developing gut of mice. Overexpression of the PDGFR-alpha gene in gastrointestinal stromal tumors (GISTs) indicated that parts of tumor cells originated from PDGFR-alpha-positive cells, but a more detailed distribution of PDGFR-alpha and possible role in the adult mammalian gut are still unclear. In the present study, we examined the expression of both PDGF-AA and its receptor PDGFR-alpha in the gastrointestinal (GI) tract of adult guinea pigs using western blotting and immunohistochemistry. PDGF-AA-immunoreactive cells were mainly distributed in the mucosal epithelium of the stomach, small intestine, and large intestine. Only a few PDGF-AA-positive cells were seen in the longitudinal muscle layer of the large intestine. In contrast, PDGFR-alpha-positive cells were widely distributed throughout the GI tract, including the lamina propria, muscular layer, and subserosa. Double staining showed that the distribution of the PDGFR-alpha-positive cells in the muscular layer were similar to those of the interstitial cells of Cajal (ICCs), and they were associated with ICCs and enteric nerves, but no double-labeled cells were observed by anti-PDGFR or Kit antibody. It was noted that PDGFR-alpha-positive cells were also stained with a vimentin monoclonal antibody. Based on the double staining and morphological features, we consider the PDGFR-alpha-positive cells belong to a subtype of fibroblast. Our results not only provide a roadmap for understanding the function of the PDGF/PDGFR signaling pathway in both normal adult mammals and during gut injury and repair but also might help in understanding the growth and development of GISTs in the clinic.