Objective: To determine the role of the rho-associated kinase (ROK) for the regulation of forearm blood flow (FBF) and unmask a potential role of ROK for regulation of endothelium-derived nitric oxide (NO). Moreover, the effect of fasudil on the constrictor response to endothelin-1 was recorded. Background: Phosphorylation of the myosin light chain (MLC) determines the calcium sensitivity of the contractile apparatus. MLC phosphorylation depends on the activity of the MLC kinase and the MLC phosphatase. The latter enzyme is inhibited through phosphorylation by ROK. ROK has been suggested to inhibit NO generation, possibly via inhibition of the AKT pathway. Methods: The effect of intra-arterial infusion of the ROK inhibitor fasudil on FBF in 12 healthy volunteers was studied by venous-occlusion plethysmography. To unmask the role of NO fasudil was infused during NO-clamp. Results: Fasudil increased FBF dose-dependently from 2.34 ± 0.21 to 6.96 ± 0.93 ml (100 ml forearm volume)^-1 at 80 µg min^-1 (P < 0.001). At 1600 µg min^-1 fasudil reduced systemic blood pressure while increasing heart rate. Fasudil abolished the vasoconstrictor effect of endothelin^-1. The vascular response to fasudil (80 µmol min-1) was blunted during NO-clamp (104% ± 18 vs 244% ± 48 for NO-clamp + fasudil vs fasudil alone; baseline = 0%, P < 0.05). Conclusions: 1) Basal peripheral and systemic vascular tone depends on ROK. 2) A significant portion of fasudil-induced vasodilation is mediated by NO suggesting that vascular bioavailable NO is negatively regulated by ROK. 3) The constrictor response to ET involves ROK activation.