The recent identification in Heloderma horridum venom of exendin-3, a new member of the glucagon superfamily that acts as a pancreatic secretagogue, prompted a search for a similar peptide in Heloderma suspectum venom. An amino acid sequencing assay for peptides containing an amino-terminal histidine residue (His1) was used to isolate a 39-amino acid peptide, exendin-4, from H. suspectum venom. Exendin-4 differs from exendin-3 by two amino acid substitutions, Gly2-Glu3 in place of Ser2-Asp3, but is otherwise identical. The structural differences make exendin-4 distinct from exendin-3 in its bioactivity. In dispersed acini from guinea pig pancreas, natural and synthetic exendin-4 stimulate a monophasic increase in cAMP beginning at 100 pM that plateaus at 10 nM. The exendin-4-induced increase in cAMP is inhibited progressively by increasing concentrations of the exendin receptor antagonist, exendin-(9-39) amide. Unlike exendin-3, exendin-4 does not stimulate a second rise in acinar cAMP at concentrations greater than 100 nM, does not stimulate amylase release, and does not inhibit the binding of radiolabeled vasoactive intestinal peptide to acini. This indicates that in dispersed pancreatic acini, exendin-4 interacts only with the recently described exendin receptor.