Binding studies were performed on rat pancreatic dispersed acini with (--)-[3H]QNB, in parallel with secretory studies to establish that the muscarinic receptors characterized are involved in enzyme secretion. Scatchard analysis of [3H]QNB saturation isotherm gave an apparent KD value of 3.09 X 10(-10) M with binding capacities of 2605 fmol/mg of DNA. The true KD calculated from the ratio of the kinetic parameters was 64 pM. The receptor population can be divided into two classes for the agonist binding sites: a population of high affinity sites and one of low affinity sites. With carbamylcholine, maximal stimulation of amylase release occurred with 40% receptor occupation while occupation of the remaining 60% caused a progressive decrease in stimulation of amylase release. Moreover, the dissociation constant of the high affinity sites for carbamylcholine (1.78 X 10(-6) M) is very close to the ED50 of carbamylcholine (5.81 X 10(-7) M) found for the stimulation of enzyme secretion. These data suggest that the physiological response of the pancreatic acini to a muscarinic agonist would involve the high affinity site population.