Many biologically active peptides exist in multiple molecular forms, but the functional significance of regions outside the region of bioactivity is unknown. The biological and immunological data presented in this study indicate that cholecystokinin-58 (CCK-58), unlike other forms of cholecystokinin, has structure that influences its bioactivity. CCK-58 was purified from acid extracts of canine intestinal mucosa until a single absorbance peak was obtained during reverse-phase chromatography. Amino acid analysis precisely determined the peptide concentrations of purified CCK-58 and synthetic CCK-8. Our hypothesis was that if the amino terminus of CCK-58 influences its bioactivity then its activity would be modified when this region was removed from the peptide. To evaluate the importance of the amino terminus of CCK-58 to influence its biological activity, the abilities of CCK-58 and CCK-8 to release amylase from pancreatic acini were compared before and after tryptic digestion. Tryptic digestion of CCK-58 decreased the half-maximal stimulation (EC50) for amylase release from 96 to 28 pM. The EC50 for digested CCK-58 was similar to that for CCK-8 (17 pM). These results suggest that CCK-58 has a structure that shields its bioactive carboxyl terminus. This is further supported by the finding that carboxyl fragments generated from CCK-58 by trypsin or by partial acid hydrolysis were greater than twofold more immunoreactive than the intact CCK-58. The diminished activity of CCK-58 SK shields the carboxyl terminus, which is important to its biological and immunological activities.