Small G protein Rho and its most studied effectors, ROCK I and ROCK II, are involved in several cellular fuctions including smooth muscle and non-muscle cell contractions, cell migration and apoptosis. Activation of ROCK I by caspase-3 and activation of ROCK II by granzyme B are essential for membrane blebbing in the execution phase of apoptosis. In contrast, it has been demonstrated that Rho signaling is critical for blebbing developed after serum removal. As it was shown by us previously, ouabain induces membrane blebbing and proteolitic cleavage of ROCK I and ROCK II via caspases in human umbilical endothelial cells. However, caspase inhibitors do not prevent ouabain-induced blebs. Ouabain induces concentration-dependent cell death and membrane blebbing in endothelial cells. The aim of this study was to identify the possible role of Rho in ouabain-induced membrane blebbing. Pretreatment of endothelial cells with a Rho inhibitor CT04 did not inhibit the ouabain-induced cell death but prevented the development of bleb formation. These results indicate that bleb formation is dependent on Rho activity in ouabain-induced cell death in HUVECs. Taken together, these results suggest that the mechanism of membrane bleb formation might be different depending on cell type and cell death-stimuli.