The endothelium is a confluent monolayer, lining the inner surface of blood vessels which acts as the main local regulator of vascular wall homeostasis. Binding of inflammatory mediators to G-protein coupled receptors increases endothelial cells permeability by increasing intracellular Ca²⁺ concentration. Several studies have shown that RhoA/Rho-kinase plays a key role in modulation of barrier properties of cultured endothelial cells and intact microvessels [W-S. Beata et al, 1998: C.M. Joes et al, 1999: R.H. Adamson et al, 2002]. Therefore we investigated role of Rho-A and its main effector, Rhokinase in control of calcium signaling in intact endothelial cells of rat tail artery using immunohistochemistry and confocal imaging. Rats were humanely killed under CO₂ anaesthesia; their tail removed from the ventral grove, cleaned of fat and loaded with Fluo-4 AM (Molecular Probes, 15um) with pluronic. Confocal imaging was done using Nipkow disc based confocal imaging system (Ultraview Perkin Elmer, UK). Minimum of 3 animals were used in each set of experiments. We have found that in endothelium of rat tail artery ROK-α but no ROK-β was expressed. Carbachol a muscurinic receptor agonist was used in our study to stimulate the intact endothelial cells. Application of carbachol (0.1uM, 1uM, 10uM) to intact endothelial cells produced a calcium transient which consisted of two components: initial fast – dependent on Ca²⁺ release and subsequent, sustained dependent on Ca²⁺ entry. Sustained component of CCh induced Ca²⁺ transient was 41 ± 0.7 of the peak taken for 100% (n= 372cells, 7vessels). The frequency of oscillation ranges from 0.05 to 0.3 Hz (n=372cells, 7vessels). Inhibition of Rho-A by C3-transferase (1µg/ml) or Rho-kinase by H-1152 (200 nM) reduced the initial fast component of CCh induced Ca²⁺ transient to 55±1.5 (n=134 cells, 3 vessels) and 52±2.1 (n=136 cells, 3 vessels) of the peak, respectively and either fully abolished or significantly decreased the amplitude and the duration of the Ca²⁺ oscillations while the amplitude of the sustained component expressed as a percentage of peak was 65±1.8% (n=134cells, 3vessels) and 29±1.6% (n=136cells, 3vessels), respectively. Taken together, the data obtained suggest that Rho-A/ROK pathway is involved in control of calcium signaling induced by CCh in intact endothelium of large conduit arteries.