Abstract Very few studies have described briefly the spatial and temporal characteristics of Ca2+ signaling using ATP, bradykinin, histamine and thrombin in cultured endothelium. However so far, the spatial and temporal characteristics in intact endothelium have not been elucidated. The aim of this work was to increase the understanding of characteristics of Ca2+ signaling in endothelial cells using real time confocal imaging of Fluo-4 loaded intact rat tail arteries. Of all the three agonist used i.e. histamine, bradykinin and carbachol; only CCh produced strong dose dependent stimulant action on the endothelial cells of intact rat tail artery. Local endothelial heterogeneity was observed in fresh intact cells in response to the three agonist used which was absent upon culture. In agreement with the previous studies, each of the three agonists produced complex Ca2+ signaling consisting of initial fast followed by sustained component with Ca2+ oscillations superimposing it. The data supports the following conclusions: (i) Ca2+ puffs were evoked in 18% and 14% of cells by low concentrations of CCh (0.1 μM and 1 μM), respectively. The average diameter and width of Ca2+ puffs initiation sites was 1.9 ± 0.4 μm, 1.6 ± 0.1 μm, respectively. Length varied between 2 and 6 μm (n = 25 vessels). (ii) ECs responding with Ca2+ waves and oscillations were dependent on concentration of CCh: 0.1 μM (20%), 1 μM (65%) and 10 μM (99%). Speed of wave propagation and frequency of Ca2+ oscillations varied among different cells even at the same agonist concentration. (iii) Ca2+ waves were regenerative; initiated by one or both ends of the cell and propagated across the cell at a speed of 14–44 μm/s (iv) With increasing concentrations of CCh (0.1–10 μM), the frequency of Ca2+ oscillations ranged from 0.02 to 0.3 Hz with optimal frequency being about 0.2 ± 0.02 Hz (n = 15 vessels). In conclusions, this data shows the morphology, heterogeneity and detailed characteristics of Ca2+ signal in intact endothelial cells.