Important functions of the vascular endothelium, including permeability, production of antithrombotic factors, and control of vascular tone, are regulated by changes in intracellular Ca²⁺. The molecular identities and regulation of Ca²⁺ influx channels in the endothelium are incompletely understood, in part because of experimental difficulties associated with application of patch-clamp electrophysiology to native endothelial cells. However, advances in confocal and total internal reflection fluorescence microscopy and the development of fast, high affinity Ca²⁺-binding fluorophores have recently allowed for direct visualization and characterization of single channel transient receptor potential (TRP) channel Ca²⁺ influx events in endothelial cells. These events, called "TRP channel Ca²⁺ sparklets," have been optically recorded from primary endothelial cells and the intact endothelium, and the biophysical properties and fundamental significance of these Ca²⁺ signals in vasomotor regulation have been characterized. This review will first briefly discuss the role of endothelial cell TRP channel Ca²⁺ influx in endothelium-dependent vasodilation, will describe improved methods for recording unitary TRP channel activity using optical methods, and will highlight discoveries regarding the regulation and physiological significance of TRPV4 Ca²⁺ sparklets in the vascular endothelium enabled by this new technology. Perspectives on the potential use of these techniques to evaluate changes in TRP channel Ca²⁺ influx activity associated with endothelial dysfunction are offered.