To study the mechanism by which endothelial cells respond to mechanical forces, we used digital fluorescence microscopy to measure changes in intracellular Ca2+ concentration ([Ca2+]i) in primary cultures of bovine aortic endothelial cells in response to mechanical stimulation. Before stimulation, [Ca2+]i was stable (approximately 50–75 nM). When an individual cell within the monolayer was mechanically stimulated with a microprobe, [Ca2+]i increased in the stimulated cell and spread in the form of a wave from the site of contact to the cell edges. After a delay of approximately 1 s, nonstimulated adjacent cells showed a similar spreading rise in [Ca2+]i. This outwardly radiating [Ca2+]i wave involved progressively more distal cells to a radius of 4–6 cells. The time delay before the wave appeared in adjacent cells increased, and peak [Ca2+]i in each cell decreased with distance from the stimulated cell. In the absence of extracellular Ca2+, there was no increase in [Ca2+]i in the stimulated cell, yet a wave of increased [Ca2+]i occurred in neighboring cells as if communicated from the stimulated cell. These results indicate that endothelial cell mechanosensitivity results in increases in [Ca2+]i and that the temporospatial dynamics of intercellular Ca2+ signaling are mediated by a diffusible substance other than Ca2+.