Diamide is a membrane-permeable, thiol-oxidizing agent that rapidly and reversibly oxidizes glutathione to GSSG and promotes formation of protein-glutathione mixed disulfides. In the present study, the acute effect of diamide on free cytosolic Ca²⁺ concentration ([Ca²⁺]_i) was examined in fura-2-loaded bovine aortic endothelial cells. At low concentrations (50, 100 μM), diamide reversibly increased spontaneous, asynchronous Ca²⁺ oscillations, whereas, at higher concentrations (250, 500 μM), diamide caused an immediate synchronized Ca²⁺ oscillation in essentially all cells of the monolayer, followed by a time-dependent rise in basal [Ca²⁺]_i. The effects of diamide on [Ca²⁺]_i dynamics were independent of extracellular Ca²⁺. Inhibition of phospholipase C by U-73122 prevented the observed changes in [Ca²⁺]_i. Additionally, the diamide-induced oscillations, but not the rise in basal [Ca²⁺]_i, were blocked by inhibition of the inositol-1,4,5-trisphosphate (IP₃) receptor (IP₃R) by 2-aminoethyl diphenyl borate. However, diamide failed to alter the plasmalemmal distribution of a green fluorescent protein-tagged phosphatidylinositol-4,5-bisphosphate binding protein, demonstrating that diamide does not activate phospholipase C. Inhibition of glutathione reductase by N,N′-bis(2-chloroethyl)-N-nitrosourea or depletion of glutathione by l-buthionine-sulfoximine enhanced the effects of diamide, which, under these conditions, could only be reversed by addition of dithiothreitol to the wash buffer. Biochemical assays showed that both the IP₃R and the plasmalemmal Ca²⁺-ATPase pump could be reversibly glutathionylated in response to diamide. These results demonstrate that diamide promotes Ca²⁺ release from IP₃-sensitive internal Ca²⁺ stores and elevates basal [Ca²⁺]_i in the absence of extracellular Ca²⁺, effects that may be related to a diamide-induced glutathionylation of the IP₃R and the plasmalemmal Ca²⁺-ATPase Ca²⁺ pump, respectively.