Previous studies indicated that the peptide substance P (SP) causes Cl--dependent secretion in animal colonic mucosa. We investigated the effects of SP in human colonic mucosa mounted in Ussing chamber. Drugs for pharmacological characterization of SP-induced responses were applied 30 min before SP. Serosal, but not luminal, administration of SP (10(-8) to 10(-6) M) induced a rapid, monophasic concentration and Cl--dependent, bumetanide-sensitive short-circuit current (Isc) increase, which was inhibited by the SP neurokinin 1 (NK1)-receptor antagonist CP-96345, the neuronal blocker TTX, the mast cell stabilizer lodoxamide, the histamine 1-receptor antagonist pyrilamine, and the PG synthesis inhibitor indomethacin. SP caused TTX- and lodoxamide-sensitive histamine release from colonic mucosa. Two-photon microscopy revealed NK1 (SP)-receptor immunoreactivity on nerve cells. The tyrosine kinase inhibitor genistein concentration dependently blocked SP-induced Isc increase without impairing forskolin- and carbachol-mediated Isc increase. We conclude that SP stimulates Cl--dependent secretion in human colon by a pathway(s) involving mucosal nerves, mast cells, and the mast cell product histamine. Our results also indicate that tyrosine kinases may be involved in this SP-induced response.