Endothelial cells occupy a unique position in the vascular regulatory environment, where they play a key role in translating neurohumoral and mechanical inputs into vasomodulatory outputs. A feature that is central to the function of endothelial cells, and represents a point of convergence of endothelial cell pathways that ultimately play a role in modulating smooth muscle tone, is elevated intracellular calcium (Ca2+). Endothelial Ca2+ levels are increased through two mechanisms: influx of extracellular Ca2+ and release of Ca2+ from intracellular endoplasmic reticulum (ER) stores. Although it is clear that inositol trisphosphate (IP3) receptors (IP3Rs) mediate Ca2+ release from the ER into the cytoplasm, the pathways that mediate the entry of extracellular Ca2+—a major contributor to changes in endothelial cell Ca2+—have remained elusive. We have recently identified IP3R-mediated, stationary Ca2+ release events (“Ca2+ pulsars”) at the endothelial projections to the smooth muscle (Ledoux et al., PNAS, 2008). The goal of the current study was to identify and characterize Ca2+ entry pathways in native endothelium, with a focus on transient receptor potential vanilloid 4 (TRPV4) channels. We used third-order mesenteric arteries (MAs) from mice that express fluorescent Ca2+ biosensor (GCaMP2) selectively in endothelial cells (ECs). TRPV4 agonist GSK1016790A (GSK, 10 nM) caused an EC-dependent dilation of pressurized MAs, which was prevented by TRPV4 antagonists, HC067047 (1 μM) and ruthenium red (5 μM) and by inhibition of EC intermediate-conductance, Ca2+-sensitive potassium (IK) channels. High speed confocal Ca2+ imaging of slit-open MAs revealed local step-wise increases in fluorescence to TRPV4 agonists (GSK, 4α-PDD, 11,12-EET), which were inhibited by TRPV4 antagonists and did not occur in the absence of external calcium or in TRPV4-/- ECs. Quantal analysis of these local Ca2+ signals (“TRPV4 Ca2+ sparklets”)(all points histograms) indicated a multimodal distribution with a mean quantal level of 0.19 (ΔF/F0). TRPV4 agonists and antagonists affected sparklet activity but not amplitude. Changing the Ca2+ electrochemical gradient by elevating external calcium from 2 to 10 mM increased the quantal level by 53%, or by membrane depolarization with 100 mM potassium decreased the quantal level by about 48%. Based on our diameter measurements, Ca2+ entry through EC TRPV4 channels could cause vasodilatation through activation of EC IK channels. We examined membrane currents in freshly isolated ECs from the same arteries, using the patch clamp technique. ECs exhibit IK and SK channel currents, with a ratio of maximal current densities of 6/1. TRPV4 agonists lead to the maximal activation of IK and SK channel currents, as measured in the perforated patch configuration. In conclusion, our results indicate that TRPV4 Ca2+ sparklets represent calcium influx through single TRPV4 channels, and this local Ca2+ can cause vasodilatation through activation of EC IK channels. Supported by AHA-PHA 10POST3690006 (SKS) and NIH HL044455, HL098243, HL095488 (MTN).