In dispersed acini from rat pancreas, binding of 125I-labeled vasoactive intestinal peptide and 125I-labeled secretin was relatively rapid, reversible, saturable, and temperature dependent. The rate of dissociation of bound 125I-labeled peptide was not a function of the concentration of free vasoactive intestinal peptide or secretin, indicating that the apparent affinities of these labeled peptides for their binding sites do not depend on the extent of receptor occupation. Four classes of receptors are required to account for the actions of vasoactive intestinal peptide and secretin on enzyme secretion, cellular cAMP, and binding of 125I-vasoactive intestinal peptide and 125I-secretin. One class has a high affinity for vasoactive intestinal peptide, and occupation of this class of receptors causes increased cellular cAMP and stimulation of amylase secretion. A second class has a low affinity for vasoactive intestinal peptide and for secretin, and occupation of these receptors does not cause changes in cAMP or amylase secretion. A third class of receptors has a high affinity for secretin, and occupation of these receptors causes increased cAMP and stimulation of amylase secretion. A fourth class of receptors has a low affinity for secretin, and occupation of these receptors causes stimulation of amylase secretion by a non-cAMP-mediated mechanism.