1. The present study was performed to determine the role of Rho-Rho kinase signalling pathway in smooth muscle cells from both healthy and varicose human saphenous vein. 2. The Rho kinase inhibitor Y-27632 inhibited the noradrenaline (NA)-induced contraction in human saphenous veins with IC(50) corresponding to 0.5 microM and 10.9 microM in control and varicose veins, respectively. The maximal amplitude of the NA-induced contraction was smaller in varicose vein compared to control (1263+/-172 mg versus 1974+/-245 mg, P<0.05). 3. In beta-escin permeabilized strips, GTPgammaS induced a rise in tension that was inhibited by Y-27632. The amplitude of the GTPgammaS-induced contraction was smaller in varicose compared to control veins (23.1+/-2.4% versus 41.3+/-2.2%, P<0.002). 4. In smooth muscle cells, Y-27632 induced disassembly of both actin cytoskeleton and extracellular fibronectin matrix. In comparison to control cells, varicose vein smooth muscle cells show decreased actin cytoskeleton organization and reduction of fibronectin matrix deposition. 5. The Rho proteins Rnd1 and RhoA, and Rho kinase 1 are expressed in human saphenous veins. A 2.6 fold reduction of Rho kinase expression was found in varicose veins. 6. These results indicate that RhoA-Rho kinase mediated Ca(2+) sensitization of the contraction and regulated actin cytoskeleton and extracellular fibronectin matrix assembly in human saphenous smooth muscle. The decrease of Rho kinase expression and Rho kinase-dependent functions detected in smooth muscle from varicose veins supports a role of this signalling pathway in the functional alterations of the vein wall occurring in the course of the disease.