Cytosolic calcium and membrane potential were monitored simultaneously in quiescent human umbilical vein endothelial cells (HUVEC) exposed to vascular endothelial growth factor (VEGF)₁₆₅ using the fluorescent indicators indo-1 AM and DiSBAC₂(3), respectively. Application of VEGF₁₆₅ to cells elicits a rapid rise in cytosolic calcium followed by a slower decline toward control values. Peak calcium is associated with a slight membrane hyperpolarization; however, as calcium falls toward control, a strong depolarization develops and is sustained throughout a 10-min period of VEGF₁₆₅ stimulation. Both the VEGF₁₆₅-mediated rise in cytosolic calcium and membrane depolarization are eliminated by inhibitors of VEGFR-2, tyrosine kinase, src kinase and inositol-1,4,5 triphosphate-operated calcium channels. Calcium entry, which is initially facilitated by transient hyperpolarization, is restricted by a substantial, sustained depolarization that developed during the downstroke of the calcium spike. Inhibition of plasmalemmal calcium channels diminished the magnitude and duration of the calcium spike, suggesting that extracellular calcium influx, secondary to stores release, is a significant component of the calcium transient. Inhibition of chloride channels substantially reduced membrane depolarization. In addition, the depolarization is modulated by PI3 kinase in a ras-independent manner. In summary, intracellular calcium and membrane potential are influenced by several key signaling cascades of VEGFR-2 activation in HUVEC.--Dawson, N. S., Zawieja, D. C., Wu, M. H., Granger, H. J. Signaling pathways mediating VEGF₁₆₅-induced calcium transients and membrane depolarization in human endothelial cells.