Ca²⁺ sensitization of vascular smooth muscle (VSM) contraction involves Rho-dependent and Rho-kinase–dependent suppression of myosin phosphatase activity. We previously demonstrated that excitatory agonists in fact induce activation of RhoA in VSM. In this study, we demonstrate a novel Ca²⁺-dependent mechanism for activating RhoA in rabbit aortic VSM. High KCl-induced membrane depolarization as well as noradrenalin stimulation induced similar extents of sustained contraction in rabbit VSM. Both stimuli also induced similar extents of time-dependent, sustained increases in the amount of an active GTP-bound form of RhoA. Consistent with this, the Rho kinase inhibitors HA1077 and Y27632 inhibited both contraction and the 20-kDa myosin light chain phosphorylation induced by KCl as well as noradrenalin, with similar dose-response relations. Either removal of extracellular Ca²⁺ or the addition of a dihydropyridine Ca²⁺ channel antagonist totally abolished KCl-induced Rho stimulation and contraction. The calmodulin inhibitor W7 suppressed KCl-induced Rho activation and contraction. Ionomycin mimicked W7-sensitive Rho activation. The expression of dominant-negative N¹⁹RhoA suppressed Ca²⁺-induced Thr⁶⁹⁵ phosphorylation of the 110-kDa regulatory subunit of myosin phosphatase and phosphorylation of myosin light chain in VSM cells. Finally, either the combination of extracellular Ca²⁺ removal and depletion of the intracellular Ca²⁺ store or the addition of W7 greatly reduced noradrenalin-induced and the thromboxane A₂ analogue–induced Rho stimulation and contraction. Taken together, these results indicate the existence of the thus-far unrecognized Ca²⁺-dependent Rho stimulation mechanism in VSM. Excitatory receptor agonists are suggested to use this pathway for simulating Rho.