Vasoactive intestinal peptide (VIP), secretin, and C-terminal octapeptide of cholecystokinin (CCK-8) receptors were identified in rat pancreatic plasma membranes by the ability of these peptides to stimulate adenylate cyclase activity. The membrane preparation procedure was conducted through a series of steps including discontinuous sucrose density gradient fractionation. 5 mM beta-mercaptoethanol was added stepwise. Membrane preparations obtained stepwise were preincubated for 10 min at 25 degrees C in the presence of various concentrations of beta-mercaptoethanol or dithiothreitol before assaying adenylate cyclase. The use of the reducing agents exerted no effect on p[NH]ppG-, NaF-, and CCK-8- stimulated activities. By contrast, stimulation of adenylate cyclase by low VIP concentrations was specifically altered when beta-mercaptoethanol was used during tissue homogeneization at 5 degrees C. In addition, both VIP and secretin responses were highly sensitive towards a preincubation of 10 min at 25 degrees C in the presence of dithiothreitol. These results were likely to reflect alterations at the receptor level. 125I-VIP binding was, indeed, reduced after dithiothreitol preincubation, low concentrations of the thiol reagent decreasing the apparent number of high-affinity VIP receptors and higher dithiothreitol concentrations reducing the affinity of VIP receptors.