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PURPOSE To evaluate whether exposure to Rho-associated protein kinase (ROCK) inhibitor will promote human-cultured corneal endothelial cells (CECs) survival in a commercial storage medium. SETTING Edith Wolfson Medical Center, Holon, and Sheba Medical Center, Tel Hashomer, Israel. DESIGN Experimental study. METHODS Fragments of human donor corneolimbal rings were stored in commercial storage media for 1 week, half with the addition of 10 μM ROCK inhibitor (Y-27632). Evaluation of CECs for early and late apoptosis\necrosis rates was performed using anti-human CD166 antibody and flow cytometric double staining analysis of propidium iodide and Annexin V. RESULTS CECs of 6 corneolimbal rings demonstrated a reduced early apoptosis rate (4.35% ± 1.07% vs 12.18% ± 5.5%, P = .026) and a reduced late apoptosis\necrosis rate (5.5% ± 2.39% vs 9.43% ± 2.61%, P = .004) compared with control. Subsequently, the rate of apoptotic CECs expressing ROCK was significantly lower in cells exposed to ROCK inhibitor compared with cells that were not (19.01% ± 4.17 vs 30.42% ± 4.27, P < .001). CONCLUSIONS ROCK inhibitor reduced endothelial cell loss in vitro and might be used to limit or slow CEC loss in donor corneal tissue during eye banking. This might be a promising new method for promoting future graft survival.