ATP has a long-lasting vasodilatory effect, possibly due to its capability to induce a prolonged increase in the intracellular Ca2+ concentration ([Ca2+]i) in endothelial cells (EC) and activate constitutive nitric oxide synthase. However, contradictory data have been reported regarding the time course of ATP-evoked Ca2+ signals in in situ EC. In particular, short-duration Ca2+ signals have been reported, which might be thought to be unable to sustain a prolonged, NO-induced vasodilation. The current experiments were therefore performed in in situ EC of rat aorta in order to more fully define the time course of ATP-evoked Ca2+ signals. 20 µM ATP evoked a short-lasting Ca2+ signal. However, medium stirring, high agonist concentrations, inhibition of ectonucleotidases and application of a poorly hydrolyzable agonist evoked long-lasting Ca2+ signals (up to 20 min at 37°C). These studies suggest that ATP is able to sustain a prolonged [Ca2+]i increase, unless ectonucleotidase activity reduces the agonist concentration near the EC surface to subthreshold values, quickly cutting the Ca2+ signal. Furthermore, the amplitude of the long-lasting phase of the Ca2+ signal depended on the balance between agonist degradation by ectonucleotidases and agonist transport, by diffusion and convection, from bulk solution to the EC surface.