We investigated the structural requirements for bombesin (BB)-like peptides to stimulate amylase secretion in rat pancreatic acini and examined the responsible intracellular signal transduction pathways. The tetradecapeptide BB-(114) was a full agonist, whereas the heptapeptide BB-(814) did not evoke amylase secretion. The mammalian BB analog neuromedin C decapeptide [NMC-(514)] was as potent as BB-(114) in stimulating amylase secretion, suggesting that Gly⁵-Asn⁶-His⁷ (or Gln⁷) of the COOH-terminal decapeptide are essential amino acids for full biological activity. BB and NMC equipotently stimulated D-myo-inositol 1,4,5-trisphosphate production, which was inhibited by the phospholipase C (PLC) inhibitor U-73122. BB and NMC also stimulated protein tyrosine kinase (PTK) activities. The half-maximal effective concentration (EC₅₀) for NMC-activated PTK was 2 log units less than the EC₅₀ for BB-activated PTK. NMC was 10-34 times more potent than BB in increasing leukotriene C₄ (an index of arachidonic acid production). The production of leukotriene C₄ was inhibited by the phospholipase A₂ (PLA₂) inhibitor ONO-RS-082. NMC is structurally homologous to BB-(514) except that Gln⁷ in BB is replaced by His⁷ in NMC. Therefore, substitution of Gln⁷ for His⁷ may alter the signal transduction systems to include the PTK and PLA₂ pathways. U-73122 inhibited Ca²⁺ spiking and amylase secretion induced by NMC and BB. However, the PTK inhibitor genistein and the PLA₂ inhibitor ONO-RS-082 inhibited secretion induced by NMC but not that induced by BB. In contrast to nonmammalian BB receptors, which primarily use the PLC pathway, the rat BB receptor is linked to three different signal transduction systems: PLC, PTK, and PLA₂ pathways.