Endothelial intracellular calcium ([Ca²⁺]_i) plays an important role in the function of the juxtaglomerular vasculature. The present studies aimed to identify the existence and molecular elements of an endothelial calcium wave in cultured glomerular endothelial cells (GENC). GENCs on glass coverslips were loaded with Fluo-4/Fura red, and ratiometric [Ca²⁺]_i imaging was performed using fluorescence confocal microscopy. Mechanical stimulation of a single GENC caused a nine-fold increase in [Ca²⁺]_i, which propagated from cell to cell throughout the monolayer (7.9 ± 0.3 μm/s) in a regenerative manner (without decrement of amplitude, kinetics, and speed) over distances >400 μm. Inhibition of voltage-dependent calcium channels with nifedipine had no effect on the above parameters, but the removal of extracellular calcium reduced Δ[Ca²⁺]_i by 50%. Importantly, the gap junction uncoupler α-glycyrrhetinic acid or knockdown of connexin 40 (Cx40) by transfecting GENCs with Cx40 short interfering RNA (siRNA) almost completely eliminated Δ[Ca²⁺]_i and the calcium wave. Breakdown of extracellular ATP using a scavenger cocktail (apyrase and hexokinase) or nonselective inhibition of purinergic P2 receptors with suramin, had similar blocking effects. Scraping cells off along a line eliminated physical contact between cells but did not effect calcium wave propagation. Using an ATP biosensor technique, we detected a significant elevation in extracellular ATP (Δ = 76 ± 2 μM) during calcium wave propagation, which was abolished by Cx40 siRNA treatment (Δ = 6 ± 1 μM). These studies suggest that connexin 40 hemichannels and extracellular ATP are key molecular elements of the glomerular endothelial calcium wave, which may serve important juxtaglomerular functions.