In the present study the ability of two classes of substance P (SP) antagonists to affect SP- and bombesin (Bn)-stimulated amylase release, as well as binding of 125I-Bolton-Hunter (BH)-SP and 125I-[Tyr4]Bn, was studied to determine their mechanism of action and the relationship between inhibition of the action of SP and the action of Bn. Four SP analogues ([D-Pro2,D-Phe7,D-Trp9]SP, [D-Pro2,D-Trp7,9]SP, [D-Arg1,D-Pro2,D-Trp7,9,Leu11]SP, and [D-Arg1,D-Trp7,9,Leu11]SP) and two SP-(4-11) analogues ([D-Pro4,D-Trp7,9]SP-(4-11) and [D-Pro4,D-Trp7,9,10]SP-(4-11)] did not stimulate amylase release when present alone but inhibited SP- and Bn-stimulated amylase release. For each SP analogue, inhibition was specific for peptides that stimulate release by interacting with SP or Bn receptors, whereas both SP-(4-11) analogues also inhibited cholecystokinin-stimulated amylase release. Each SP analogue's inhibition of Bn-stimulated amylase release was reversible. Each analogue inhibited binding of 125I-BH-SP with the same potency as it inhibited SP-stimulated amylase release and 125I-[Tyr4]Bn binding with the same potency as it inhibited Bn-stimulated amylase. In contrast, SP itself or SP-(4-11) itself did not inhibit Bn-stimulated amylase release or binding of 125I-[Tyr4]Bn. The relative affinities of the analogues for interacting with Bn and SP receptors differed. None of the analogues increased the dissociation of bound 125I-BH-SP or 125I-[Tyr4]Bn.(ABSTRACT TRUNCATED AT 250 WORDS)