In this study, we attempted to clarify the mechanism of tachykinin-induced motor response in isolated smooth muscle preparations of the human colon. Fresh specimens of normal colon were obtained from patients suffering from colonic cancer. Using mucosa-free smooth muscle strips, smooth muscle tension with circular direction was monitored isometrically. Substance P (SP), neurokinin A (NKA), and neurokinin B (NKB) produced marked contraction. All of these contractions were inhibited by saredutant, a selective NK₂-R antagonist, but not by CP122721, a selective NK₁-R antagonist or talnetant, a selective NK₃-R antagonist. βAla⁸-NKA(4-10) induced concentration-dependent contraction similar to NKA, but Sar⁹-Met¹¹-SP and Met-Phe⁷-NKB did not cause marked contraction. Colonic contraction induced by βAla⁸-NKA(4-10) was completely blocked by saredutant, but not by atropine. Tetrodotoxin or N^G-nitro-L-arginine methyl ester pretreatment significantly enhanced βAla⁸-NKA(4-10)–induced contraction. Immunohistochemical analysis showed that the NK₂-R was expressed on the smooth muscle layers and myenteric plexus where it was also co-expressed with neuronal nitric oxide synthase in the myenteric plexus. These results suggest that the NK₂-R is a major contributor to tachykinin-induced smooth muscle contraction in human colon and that the NK₂-R–mediated response consists of an excitatory component via direct action on the smooth muscle and an inhibitory component possibly via nitric oxide neurons.