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To investigate the toxicity of topical glaucoma medications using cultured stratified human corneal epithelial sheets (HCES).HCES were exposed for 30 minutes to the following glaucoma medications: 0.1% brimonidine with sodium chlorite as the preservative, 0.005% latanoprost with 0.02% benzalkonium chloride (BAC) as the preservative, and 0.5% timolol with 0.005% BAC as the preservative. Then, cell viability and barrier function were tested by the WST-1 assay and carboxyfluorescein permeability assay, respectively. After exposure to glaucoma medications, HCES were evaluated by hematoxylin and eosin staining, periodic acid–Schiff staining, scanning electron microscopy, and transmission electron microscopy.HCES exposed to brimonidine showed higher viability and better preservation of cell morphology and microvilli compared with cell sheets exposed to latanoprost or timolol. The carboxyfluorescein permeability assay demonstrated that the barrier function was preserved after HCES were exposed to timolol, but not after exposure to brimonidine or latanoprost. Transmission electron microscopy revealed widening of intercellular junctions with prominent deposits of glycogen or mucopolysaccharide (periodic acid–Schiff positive) after exposure of HCES to brimonidine.The toxicity of 0.1% brimonidine containing sodium chlorite for HCES was lower than that of ophthalmic preparations containing BAC. Reduction of the barrier function occurred after HCES were exposed to brimonidine because of widening of intercellular junctions.