Objective: In a previous study, we showed that the spread of calcium waves via gap junctions decisively contributed to the overall response of cultured HUVEC to histamine thereby affecting endothelial NO production. Here we sought to clarify whether this gap junction dependent modulation can also be found for another endothelial dilator, ATP. Moreover we studied, whether such a role for gap junctions can also be found in the endothelium of intact vessels. Methods and results: HUVECs were stimulated with 1M ATP and the resulting changes of intracellular free calcium concentrations were detected using Fura2 with a time resolution of 2-4 Hz. Location and time course of the calcium responses of individual cells in the monolayers (n=10) were studied by videomicroscopy. Initially (0-3 sec), ATP induced intracellular calcium responses only in part of the cells, which were randomly located in the monolayer. From these "pacemaker cells" a calcium wave spread into neighbouring cells resulting in a calcium rise in virtually all cells of the monolayer within 10 sec. Inhibition of gap junction coupling (GJC) by heptanol/meclofenamic acid restricted the ATP induced intracellular calcium increase to the initial pacemaker cells whereas treatment with ionomycin still led to a homogeneous calcium increase in all cells. Similar results were also found in freshly isolated mouse aortae, which were cut for in face microscopy and treated with 25M ATP. Conclusion: The ATP induced calcium rise in cultured human and native mouse aortic endothelial cells is only partly a direct receptor induced event being restricted to a few cells acting as "pacemakers". A homogenous calcium response of endothelial cells to ATP critically depends on GJC, which suggests a new modulator role of GJC on apparent endothelial sensitivity to vasodilators.