We have demonstrated that, besides his role in the calcium-sensitization of the contractile process in smooth muscle, Rho-dependent kinase (ROK) is involved in noradrenaline-activated calcium entry in rat arteries (Ghisdal,et al. 2003). The objective of the present study was to investigate the involvement of the ROK pathway in the calcium signal in vascular smooth muscle cells, by comparing the effect of the ROK inhibitor, Y-27632, in whole artery and in cultured vascular smooth muscle cells. Endothelium denuded rat aorta rings and primary cultured rat aortic smooth muscle cells were used. Cytosolic calcium concentration was measured in fura-2-loaded arteries or cells. Vasopressin was first applied in Ca2+-free solution to release intracellular Ca2+ and Ca2+ entry was activated by the addition of Ca2+ in the bath solution. ROK expression was measured by western blot analysis and immunofluorescence, and its activity was determined by measuring its downstream targets, phospho-ERM (pERM). Protein kinase C (PKC) involvement on pERM was also investigated by measuring the effect of the PKC inhibitor, Gö-6983. In isolated rat aorta, the ROK inhibitor Y-27632 completely inhibited vasopressin-evoked contraction, depressed calcium entry signal by 52 ± 6% (n=5) and inhibited ERM phosphorylation. On the contrary, in cultured aortic smooth muscle cells, Y-27632 did affect neither the calcium entry signal evoked by vasopressin nor ERM phosphorylation, although ROK expression was confirmed. On the other hand, ERM phosphorylation was inhibited by the PKC inhibitor Gö-6983 in cultured cells but not in isolated aorta. These results suggest that the mechanism of agonist-evoked Ca2+ entry is different in the whole artery compared to cultured cells, which probably lack an essential effector in the ROK pathway and might involve the PKC pathway.