Functional subtyping of the receptors responsible for the neural and nonneural effects of substance P (SP) on the canine proximal colon were studied. Selective agonists and antagonists were used with two different in vitro preparations. The mucosa contained the muscularis mucosa and attendant submucosal plexuses, whereas the epithelium was devoid of both. We obtained the following results: [Sar9,Met(O2)11]SP (Sar9SP), a neurokinin-1 receptor agonist, stimulated both preparations; senktide, a neurokinin-3 agonist, evoked a response only on the mucosal preparation; [beta-Ala8]NKA4-10, a neurokinin-2 agonist, was ineffective on both preparations; tetrodotoxin completely inhibited the mucosal responses to senktide, but the inhibitory effects on Sar9SP were only partial; CP-96,345, a neurokinin-1-selective antagonist, inhibited both epithelial and mucosal responses to Sar9SP and R487 [Trp7,beta-Ala8]NKA4-10, a neurokinin-3 antagonist, inhibited mucosal responses to senktide. Thus, the neural effects involved both neurokinin-1 and neurokinin-3 receptors, whereas the nonneural effects were mediated by neurokinin-1 receptors alone. Neurokinin-2 receptors were functionally absent.