Carassius RFamide (C-RFa) is a peptide, isolated originally from the brain of Japanese crucian carp and sharing homologies with mammalian prolactin-releasing peptides. From the physiological aspect, it is known that C-RFa has contraction-promoting action on fish intestines, but its localization in peripheral tissues is unknown. We observed the localization of C-RFa in teleost guts using an immunohistochemical technique. C-RFa-like immunoreactive (irC-RFa) sites were observed in not only the smooth muscle cells in the longitudinal muscle layer, but also in both Auerbach's and Meissner's nerve plexus in the stomach, pyloric ceca and intestine. In epithelial mucous cells, irC-RFa sites were observed in the surface mucous cells in the stomach in freshwater fish (FW), and in the goblet cells of the apical sites in the villi of the pyloric ceca and intestine in all fish. In the stomach, irC-RFa sites were found in the fundic glands of the body regions in seawater (SW) and brackish water (BW) fish, but not in FW fish. This study confirmed that one of the functions of C-RFa is the smooth muscle contraction of the longitudinal muscle layer in digestive organs. We suggest that C-RFa may have functional roles in both central and peripheral neurotransmission. In addition, it appears that the difference in C-RFa localization of SW, BW, and FW fish reflects the adaptation of the stomach function to different salinity habitats.