Stimulation of endothelial cells (ECs) with ATP evoked an increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i). In a single bovine aortic EC, the [Ca²⁺]_i rise started at a specific peripheral locus and propagated throughout the entire cell as a Ca²⁺ wave. The initiation locus was constant upon repeated stimulation with ATP or other agonists (bradykinin and thrombin). The Ca²⁺ wave was unaffected by the removal of extracellular Ca²⁺, demonstrating its dependence on intracellular Ca²⁺ release. Microinjection of heparin into the cell inhibited the ATP-induced Ca²⁺ responses, indicating that the Ca²⁺ wave is at least partly mediated by the inositol 1,4,5-trisphosphate receptor. Immunofluorescence staining revealed that caveolin, a marker protein for caveolae, is distributed heterogeneously in the cell and that Ca²⁺ waves preferentially originate at caveolin-rich cell edges. In contrast to caveolin, internalized transferrin and subunits of the clathrin-associated adaptor complexes such as adaptor protein-1 and -2 were diffusely distributed. Disruption of microtubules by Colcemid led to redistribution of caveolin away from the edges into the perinuclear center of the cell, and the ATP-induced [Ca²⁺]_i increase was initiated on the rim of the centralized caveolin. Thus, caveolae may be involved in the initiation of ATP-induced Ca²⁺ waves in ECs.