Somatostatin and vasoactive intestinal peptide were measured by radioimmunoassay in acetic acid extracts of tissue samples from the digestive tracts of seven adult cats. The concentration of immunoreactive somatostatin in antral mucosa (2926 +/- 978 pmol/g wet weight, mean +/- SE) was far above the concentrations in the head of the pancreas (442 +/- 94), distal duodenum (377 +/- 91), and ileum (355 +/- 48). Large amounts of immunoreactive vasoactive intestinal peptide were recovered from both mucosal and muscular layers of gastrointestinal tract; the highest values were observed in the muscular layer of the cecum (579 +/- 82 pmol/g) and the mucosa of the right colon (564 +/- 244). The mucosal/muscular layer ratio of vasoactive intestinal peptide concentrations increased caudally from 0.20 in the esophagus to over 1 in the ileum and colon. Upon gel filtration on G-50 Sephadex, somatostatin in the antrum, duodenum, and pancreas were eluted as a predominant peak in the volume of the tetradecapeptide, but somatostatin from the ileum and cecum was associated with a major faster component. Vasoactive intestinal peptide in the muscular layer of gastrointestinal tract and in mucosa of antrum and duodenum consisted essentially of a single form, which coeluted with the purified porcine peptide; a slower component was detected in the mucosa of the ileum and cecum. These reults indicate molecular heterogeneity of both immunoreactive somatostatin amd vasoactive intestinal peptide in the digestive tract of cats, and suggest that the variable ratio of the different molecular forms of each peptide along the gastrointestinal tract may reflect regional specificity of biologic effects and metabolism.