The critical association of connective tissue growth factor (CTGF), which is thought to be one of the downstream mediators of transforming growth factor-beta (TGF-beta), with vitreoretinal diseases remains to be clarified. In the current study, we first demonstrated the correlation between the concentrations of TGF-beta2 as well as CTGF in the vitreous and CTGF gene regulation in cultured hyalocytes. Concentrations of TGF-beta2 and CTGF in the vitreous from patients with proliferative vitreoretinal diseases were significantly higher than in those with nonproliferative diseases, and there was a positive correlation between their concentrations (r = 0.320, P < 0.01). Cultured hyalocytes expressed CTGF mRNA, which was enhanced in the presence of TGF-beta2, associated with nuclear accumulation of Smad4. TGF-beta2-dependent Smad4 translocation and CTGF gene expression were mediated through Rho kinase and at least partially via p38 mitogen-activated protein kinase. Finally, fasudil, a Rho kinase inhibitor already in clinical use, inhibited both Smad4 translocation and CTGF gene expression. In conclusion, combined effects of TGF-beta2 and CTGF appear to be involved in the pathogenesis of proliferative vitreoretinal diseases. Hyalocytes may be a possible source of CTGF and thus might play a role in vitreoretinal interface diseases. Furthermore, Rho kinase inhibitors might have therapeutic potential to control fibrotic disorders in the eye.