An evolutionary basis for the distinct forms of cholecystokinin (CCK) receptors in the mammalian brain and pancreas was examined. The brains and pancreases of ratfish, frog, snake, and chicken contained saturable, high-affinity binding sites for iodinated porcine CCK-33. In the ectothermic species, the brain and pancreas CCK receptors exhibited nearly the same relative specificities for various CCKs and gastrins. Sulfated CCK-8 and sulfated gastrin-17 were the most potent while their nonsulfated analogs and gastrin-4 were less potent. By contrast, in the chicken, the specificities of brain and pancreas CCK receptors closely resembled their mammalian counterparts. We conclude that brain and pancreas CCK receptors with new specificities for binding CCKs and gastrins evolved at the level of the divergence of endotherms (birds and mammals) from reptiles. We propose that the prior evolution of gastrin provided the selection pressure for these changes. The endotherm pancreas receptor arose in evolution by narrowing its requirement for the position of a sulfated tyrosine residue in its ligands from either the sixth or seventh position from the carboxyl terminus to the seventh position only. The endotherm brain receptor arose in evolution by losing its requirement for a sulfated ligand and by transferring its high-affinity binding domain from the tyrosine residue in the carboxyl termini of CCK and gastrin to the carboxyl-terminal tetrapeptide active site common to CCKs and gastrins.