Substance P (SP) is a potent endothelium-dependent vasodilator, and in porcine coronary arterioles the vasodilatory action of SP appears to be mediated entirely by nitric oxide. We tested the hypothesis that SP induces hyperpolarization in porcine coronary artery endothelial cells (PCAECs) by activating Ca(2+)-activated K+ (KCa) channels. With a bath Ca2+ concentration ([Ca2+]) of 1 mM, 10 nM SP elicited an increase in cytosolic [Ca2+] ([Ca2+]i) from a baseline of 25 +/- 4 nM to a peak of 808 +/- 120 nM, followed by a slowly declining plateau phase, which was absent in Ca(2+)-free bath and was abolished by addition of extracellular lanthanum or nickel. Whole cell current-clamp recordings revealed that the time course of SP-induced [Ca2+]i increases correlated closely with membrane hyperpolarization from an average resting potential of -42 +/- 2 to a peak of -79 +/- 2 mV. Under voltage clamp, SP stimulated whole cell currents with reversal potentials strongly dependent on extracellular K+ concentration. In 62% of patches tested, single-channel recordings revealed an intermediate-conductance K+ channel with activation highly correlated with the SP-induced [Ca2+]i increase. These results suggest that, in PCAECs, SP induces Ca2+ release from stores along with Ca2+ influx which activate a KCa channel leading to hyperpolarization. This may increase the driving force for Ca2+ entry and thus modulate endothelium-derived nitric oxide release.