Specific binding sites for secretin have been identified in rat fundic membranes, using 125I-secretin. The binding was saturable, reversible, time and temperature dependent. Optimal pH for binding was around 7–7.5. Scatchard plots were compatible with the existence of 2 classes of receptors; the first class with a high affinity for secretin (apparent K d of 4×10 −10 M ) and a low binding capacity (150 fmol per mg membrane protein, i.e., 4,500 high affinity sites/cell) and a class of receptors with a lower affinity (K d of 3×10 −9 M ) and a higher binding capacity (580 fmol per mg membrane protein, i.e., 17,400 sites/cell). Glucagon, gastric inhibitory polypeptide and somatostatin had no effect on secretin binding. In contrast, VIP inhibited 125I-secretin binding and stimulated adenylate cyclase activity, in both cases at a 200-times lower potency than secretin (ID 50 and Ka=2×10 −7 M VIP). The properties of these secretin receptors strongly support the concept that secretin acts as a regulatory peptide on the rat gastric epithelium.