Rho kinase (ROCK) is a downstream effector of Rho family GTPases, and two highly homologous isoforms, ROCK1 and ROCK2, are similarly inhibited by the widely used pharmacologic inhibitors. In endothelial cells (ECs), activation of ROCK regulates myosin L chain (MLC) phosphorylation, stress fiber formation and permeability increases during inflammation. This study examined isoform-specific ROCK activation in lung ECs in vitro using human pulmonary microvascular ECs and ex vivo using freshly isolated lung ECs from mice. In unstimulated human as well as mouse lung ECs, ROCK2 activity was greater than ROCK1 activity. TNF-α stimulation induced activation of both ROCK1 and ROCK2 in cultured human ECs. Studies using lung ECs freshly isolated from mice showed that intratracheal instillation of LPS induced ROCK activation in lung ECs that was inhibited by treating animals with fasudil, a pharmacologic ROCK inhibitor, and that both ROCK1 and ROCK2 were activated. Small interference RNA targeting ROCK1 or ROCK2 was used to examine their functions in regulating MLC phosphorylation and permeability increases induced by TNF-α in human ECs. TNF-α-induced MLC phosphorylation required ROCK activation. Inhibition of ROCK1 alone was not sufficient to prevent TNF-α-induced MLC phosphorylation, whereas inhibition of ROCK2 prevented TNF-α-induced late MLC phosphorylation at 24 h. Although ROCK1 was dispensable for TNF-α-induced MLC phosphorylation, ROCK1 was required for TNF-α-induced early permeability increases. Therefore, ROCK1 and ROCK2 are both activated by TNF-α and can be functionally separated in the signaling pathways leading to TNF-α-induced MLC phosphorylation and permeability increases.