The purpose of this study is to identify the membrane transport machinery and cell signaling involved in the neurokinin A-inducible release of adenosine triphosphate (ATP) as an autocrine/paracrine signal from cultured guinea-pig taenia coli (T. coli) smooth muscle cells (SMCs). ATP release evoked by neurokinin A was inhibited by L-659877, a NK(2)-receptor antagonist; by modulators for Ins(1,4,5)P(3)-sensitive Ca(2+)-signaling, U-73122, thapsigargin, and 2-APB; and by W-7, a calmodulin inhibitor, and staurosporine, a protein kinase C (PKC) inhibitor, but not by wortmannin, a phosphoinositide 3-kinase inhibitor. The evoked release was suppressed by a multidrug resistance protein (MRP)-transporter inhibitors, MK-571, indomethacin, and benzbromarone, but not by CFTR-inh 172, a CFTR-Cl(-) channel blocker, and α-glycyrrhetinic acid, a gap junction hemichannel blocker. Neurokinin A caused a marked accumulation of Ins(1,4,5)P(3) and an increase in [Ca(2+)](i) in the cultured cells. These findings suggest that stimulation of Gq/(11) protein-coupled NK(2) receptor with neurokinin A caused a substantial release of ATP from cultured T. coli SMCs and that the evoked release may be mediated by Ins(1,4,5)P(3)-sensitive Ca(2+)-signaling, further by PKC and Ca(2+)/calmodulin signals, and finally by an activation of MRP transporters as the membrane device.