Cholecystokinin (CCK)-gastrin peptides are shown to be present in the brain of all the species ranging from coelenterates to mammals. Differentiation between CCK and gastrin, presumed to be evolved from a common ancestral cerulein-like peptide, has been suggested to occur at the level of the amphibians. We examined the presence of bioassayable CCK, as determined by its potency to stimulate enzyme secretion from isolated rat pancreatic acini, in the brain of the goldfish Carassius auratus, a more primitive vertebrate than the amphibian. Among the various regions tested, the brain stem, telencephalon and spinal cord possess the highest levels of bioassayable CCK followed in decreasing order by gustatory lobes, optic tectum and duodenum. No detectable levels of CCK were found in the cerebellum. The observed bioactivity was not due to gastrin because: radioimmunoassay of the brain homogenates for gastrin revealed very low or nondetectable levels of gastrin; amylase release dose-response curves for standard CCK8 and the brain homogenate were identical; and proglumide, a competitive antagonist of CCK8, inhibited homogenate CCK-induced enzyme release with a parallel rightward shift in the dose-response curve. These observations provide evidence for the distinct presence of CCK in the brain of the goldfish suggesting that the differentiation of CCK as a distinct neuropeptide, from that of gastrin, occurs at the level of Osteichthyes (bony fish).