Gastrin-releasing peptide (GRP) and bombesin can stimulate pepsinogen release by both gastrin-dependent and -independent mechanisms. Using isolated guinea pig gastric chief cells, we determined that GRP can act directly on the guinea pig chief cell to cause pepsinogen release. GRP and bombesin stimulated a 2.5- to 3-fold increase in pepsinogen release above basal release. Substance P also stimulated a small but significant increase in pepsinogen release. No gastrin immunoreactivity was detected in the supernatants of cells stimulated with up to 1 microM GRP or bombesin or 1 mM carbachol. GRP-stimulated pepsinogen release was completely inhibited by GRP/bombesin receptor agonists as well as substance P receptor antagonist but not by antagonists to receptors for gastrin, the octapeptide of cholecystokinin (CCK-8), secretin, vasoactive intestinal peptide (VIP), or muscarinic agents. Substance P-stimulated pepsinogen release was completely inhibited by substance P receptor antagonist but not by GRP/bombesin receptor antagonists. An additive effect on pepsinogen release was seen when GRP was combined with maximally effective concentrations of adenosine 3',5'-cyclic monophosphate (cAMP)-mediated agents (VIP, secretin, 8-BrcAMP) but not with calcium-mediated agents (carbachol, CCK-8, gastrin). These results indicate that GRP can directly stimulate pepsinogen release from guinea pig chief cells by a specific GRP receptor that mobilizes intracellular calcium.