Carassius RFamide (C-RFa) is a brain-gut peptide, isolated originally from the brain of a teleost fish, Japanese crucian carp (Carassius auratus langsdorfii). It is a novel FMRFamide-related peptide sharing homologies with mammalian prolactin-related peptide. We observed the localization of C-RFa-like immunoreactivity (C-RFa-IR) in the stomach, intestine, and pancreas of Japanese butterfly ray using an immunohistochemical technique. In the whole gastrointestinal tract, C-RFa-IR was observed in smooth muscle cells, and in the Meissner's and Auerbach's plexus ganglia. Furthermore, in the stomach C-RFa-IR was also observed at the bottom of the fundic glands in both the body and pyloric region. In the intestine C-RFa-IR was observed in epithelial cells at the tip of mucosal folds. In the pancreas, C-RFa-IR was observed in islet cells. Thus, we suggest that ray C-RFa may have functional roles in both peripheral neurotransmission and endocrine function of the gastroenteropancreatic system.