The distribution and characterization of calcitonin gene-related peptide immunoreactivity in the digestive system of normal, capsaicin-treated, and littermate control rats were studied by radioimmunoassay, chromatography, and immunohistochemistry. The highest concentrations of calcitonin gene-related peptide immunoreactivity were found in the stomach (45 +/- 2.8 pmol/g wet wt, nonsecretory region; 38.7 +/- 4.4 pmol/g wet wt, secretory region) and rectum (30.9 +/- 1.6 pmol/g wet wt). Significant amounts of peptide were also found in the other regions of the gut and in the pancreas. Neonatal treatment with capsaicin, which causes a permanent degeneration of most of the small-diameter sensory neurons, reduced calcitonin gene-related peptide content by greater than 95% in the esophagus and stomach, by 60% in the pancreas, and by less than 50% in the intestine, when compared with littermate controls. Separation of extracts from the gut, pancreas, and brain by chromatography gave major peaks corresponding to the predicted rat calcitonin gene-related peptide and small unidentified peaks, which presumably arise from metabolism of the peptide. Immunohistochemical studies demonstrated that in the esophagus and stomach, calcitonin gene-related peptide immunoreactivity is restricted to nerve fibers, whereas in the intestine it is localized in both nerve fibers and enteric ganglion cells. In capsaicin-treated rats there was a virtually complete elimination of calcitonin gene-related peptide immunoreactive fibers innervating the esophagus and stomach, whereas in the small and large intestine there was a dramatic reduction and often a complete elimination of those associated with blood vessels and a slighter reduction of the nonvascular immunoreactive fibers. The results of this study indicate that calcitonin gene-related peptide immunoreactive nerve fibers innervating the rat digestive system originate from both intrinsic (enteric) and extrinsic (presumably sensory) sources and that both the intrinsic and extrinsic components appear to contain a substance that corresponds to the predicted calcitonin gene-related peptide.