The aim of the study was to assess, in intact murine thoracic aorta in vitro, the distribution of endothelial cells responsive to endothelium-dependent vasodilators ACh, ATP, bradykinin and substance P, using laser line confocal microscopy in combination with two Ca²⁺ fluorescent dyes, Fluo-4 and Fura-red. We observed that 82 ± 3% of endothelial cells responded to ATP, 33 ± 5% to Ach, whereas less than 3% of them responded to bradykinin or substance P. In order to determine whether the findings of pharmacological tests agree with confocal microscopy data, endothelium-dependent vasodilators induced relaxation was evaluated using isometric tension measurement. We show a marked correlation between a higher number of activated endothelial cells, using confocal microscopy, and a greater degree of endothelium-dependent relaxation using isometric tension measurement (p = 0.00286). Our results suggest that endothelial cells responding to endothelium-dependent vasodilators are not homogeneously distributed in intact murine thoracic aorta. This could be due to nonhomogeneous distribution of surface receptors or to differences in post-receptor coupling mechanisms.