The role of the amino acid in position 31 of cholecystokinin CCK26-33 in the recognition of central and peripheral receptors was investigated by replacement of methionine-31 by amino acids with side chains of various chemical nature. Thus, phenylalanine, alanine, glutamic acid, and ornithine and its analogue with the epsilon-amino group protected by a benzyloxycarbonyl group were introduced as X residues in Boc(Nle28,X31)-CCK27-33 since the related analogue Boc(Nle28,Nle31)-CCK27-33 was shown to be equipotent to CCK26-33. The binding properties to both mouse brain membranes and guinea pig pancreatic acini and the peripheral activities (amylase secretion and contractile potency on guinea pig ileum) were determined. Whereas the introduction of phenylalanine, alanine, or ornithine residues in position 31 led to compounds that still displayed peripheral agonist properties, the presence of a negative charge in the side chain of the amino acid in position 31 prevented the binding of the peptide to both pancreatic and brain binding sites. Introduction of Phe31 and Ala31 residues increased the specificity of the peptides for the central receptors. Interestingly, when the amine function in the side chain of the ornithine-31 was protected by a benzyloxycarbonyl group, an unusual high affinity for pancreatic binding sites was observed and the related analogue proved to be a new peripheral CCK antagonist.