Disruption of the actin cytoskeleton elicits profound changes in cell survival and function. The actin cytoskeleton is regulated in a hierarchical manner by Rho GTPases. Rho kinase, a downstream effector of RhoA, regulates the formation of stress fibers and focal adhesions. Disruption of the actin cytoskeleton causes apoptosis in airway epithelial cells. To examine further the relation of cytoskeletal integrity and apoptosis, we tested whether inhibition of Rho kinase would elicit apoptosis in airway epithelial cells. Inhibition with either Y-27632 or HA1077 induced membrane ruffling and loss of actin stress fibers, and apoptosis in airway epithelial cells that was blocked by inhibiting caspase function or by inhibiting protein synthesis. Cells over-expressing constitutively active Rho kinase, but not native Rho kinase, were resistant to Rho kinase inhibitor-induced stress fiber disruption and apoptosis. Inhibition of Rho kinase disrupted actin stress fibers but did not induce apoptosis in 3T3 cells. We demonstrate that Rho kinase inhibition induces airway epithelial cell apoptosis associated with changes in actin filament integrity. Our data suggest that Rho kinase may be a regulator of early initiation of apoptosis.