In addition to its role in artery contraction, Rho kinase (ROCK) is reported to be involved in the Ca(2+) response to vasoconstrictor agonist in rat aorta. However the signaling pathway mediated by ROCK had not been investigated so far and it was not known whether ROCK also contributed to Ca(2+) signaling in cultured vascular smooth muscle cells (VSMC), which undergo profound phenotypic changes. Our results showed that in VSMC, ROCK inhibition by Y-27632 or H-1152 had no effect on the Ca(2+) response to vasopressin, while in aorta the vasopressin-induced Ca(2+) entry was significantly decreased. The inhibition of myosin light chain kinase (MLCK) by ML-7 depressed the vasopressin-induced Ca(2+) signal in aorta but not in VSMC. The difference in ROCK sensitivity of vasopressin-induced Ca(2+) entry between aorta and VSMC was not related to an alteration of the RhoA/ROCK pathway. However, MLCK expression and activity were depressed in cultured cells compared to aorta. We concluded that the regulation of vasopressin-induced Ca(2+) entry by ROCK in aorta could involve the myosin cytoskeleton and could be prevented by the downregulation of MLCK in VSMC. These results underline the important differences in Ca(2+) regulation between whole tissue and cultured cells.