Immunoreactive GH release-inhibiting factor [somatostatin (SRIF)] was detected in hypothalamus, extrahypothalamic brain, pancreas, and stomach extracts of the rat, pigeon, tortoise, frog, teleost (cichlid), and elasmobranch (dogfish) and in the whole brain of the cyclostome (hagfish). The SRIF concentration was higher in the pancreas and gastrointestinal tract than in the hypothalamus and extrahypothalamic brain in most species. Extracts of the various tissues from the different species assayed in serial dilutions gave displacement curves parallel to those of synthetic mammalian SRIF. Cation exchange chromatography of hypothalamic extracts from the various species revealed two major immunoreactive peaks, one of which corresponds to synthetic SRIF in elution volume, the other being less basic. Affinity chromatography-purified immunoreactive SRIF from frog brain, pancreas, and stomach extracts coeluted with synthetic SRIF in high pressure liquid chromatography. The results indicate that immunoreactive SRIF in various tissues and in different vertebrates is indistinguishable and suggest that there has been no change in the molecule during at least 400 million yr of evolution