We investigated the roles of Rho-associated protein kinase (ROCK) in regulating activities such as adhesion, contraction and migration in cultured human trabecular meshwork (TM) cells. Human TM cells in culture were treated with Y-27632, a specific ROCK inhibitor. Trypan blue exclusion test and TUNEL staining showed little or no direct toxicity of Y-27632 on TM cells. By MTT assay, Y-27632 did not significantly affect the proliferation of TM cells. The cell adhesion assay showed that Y-27632 promoted the cell adhesiveness to both fibronectin and collagen type I in a dose-dependent manner. Collagen gel contraction activity of TM cells was significantly inhibited by the treatment of Y-27632 in a dose-dependent manner. The addition of Y-27632 accelerated motility of TM cells in wound healing assay. Phosphorylated LIM kinase 2 and cofilin, related to actin bundling and integrin clustering, were dephosphorylated (activated) by Y-27632. In conclusion, Y-27632 elicits profound effects on TM cell activities including adhesion, gel contraction, and cell motility. These Y-27632-induced changes of TM cells may be relevance to the physiology of the aqueous outflow system.