We prepared 125I-secretin and studied the kinetics, stoichiometry, and chemical specificity with which the labeled peptide binds to dispersed acini prepared from guinea pig pancreas. Iodinated secretin retained intrinsic biological activity in that it was as effective but 2.5-times less potent than native secretin in its ability to bind to pancreatic acini and to increase cellular cAMP. Scatchard analysis of binding of 125I-secretin indicated that each pancreatic acinar cell has approximately 93,000 binding sites, half of which are occupied by 11 nM iodinated secretin. Binding of 125I-secretin was rapid, reversible, saturable, specific, and temperature dependent. Binding of 125I-secretin was inhibited by secretin, vasoactive intestinal peptide, PHI, and Gila monster venom but not by glucagon, gastric inhibitory polypeptide, cholecystokinin, caerulein, gastrin, bovine pancreatic polypeptide, somatostatin, neurotensin, leucine-enkephalin, methionine-enkephalin, carbachol, bombesin, litorin, eledoisin, physalaemin, or substance P. With agonists (secretin, vasoactive intestinal peptide, PHI, or Gila monster venom), as well as antagonists (C-terminal fragments of secretin), there was a close correlation between their relative potencies for inhibiting binding of 125I-secretin and their relative potencies for increasing cAMP (agonists) or inhibiting the secretin-induced increase in cAMP (antagonists). For a given agonist, however, a 40-fold higher concentration was required for half-maximal inhibition of binding of 125I-secretin than was required to produce a half-maximal increase in cellular cAMP. Thus, maximal stimulation of cellular cAMP occurs when approximately one-third of the secretin receptors are occupied by an agonist.