CCK-58(ns) (non-sulfated cholecystokinin 58) has not been considered to be of biological importance because CCK-58(ns) binds poorly to the CCK-A receptor and has only been identified once in intestinal extracts. In this work, a radioimmunoassay specific for the C-terminal region of gastrin and cholecystokinin (antibody 5135) was used to monitor the purification of cholecystokinin molecular forms from canine intestinal extracts. A minor immunoreactive peak was associated with a major absorbance peak during an ion-exchange high performance chromatography step. Characterization of this minor immunoreactive peak demonstrated that it was CCK-58(ns). CCK-58(ns) is 14% as immunoreactive as sulfated CCK-8 [CCK-8(s)]. Amino acid analysis demonstrated that CCK-58(ns) was present at 50% the amount of CCK-58(s). In addition, we found that CCK-58(ns) does not potently displace a ¹²⁵I-CCK-10 analog from the CCK-A receptor in mouse pancreatic membranes, does not stimulate amylase release from isolated pancreatic acini, or stimulate pancreatic secretion in an anesthetized rat model. By contrast, CCK-58(ns) does bind to CCK-B receptors and stimulates gastric acid secretion via this receptor. The presence of CCK-58(ns) and its ability to selectively stimulate the CCK-B receptor without stimulation of the CCK-A receptor suggest that CCK-58(ns) may have unique physiological properties, especially tissues where the non-sulfated peptide can act as a paracrine or neurokine agent.