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The aim of this study was to evaluate the toxicity of intrastromally injected voriconazole on corneal endothelial cells in rabbits.In total, 32 eyes of 16 rabbits (8 eyes for each group) were divided into 4 groups according to the concentration of voriconazole [group A: 50 μg/0.1 mL (0.05%), group B: 100 μg/0.1 mL (0.1%), group C: 250 μg/0.1 mL (0.25%), or group D: 500 μg/0.1 mL (0.5%)]. Right eyes were injected intrastromally with voriconazole at concentrations of 50 μg/0.1 mL, 100 μg/0.1 mL, 250 μg/0.1 mL, or 500 μg/0.1 mL. Left eyes were injected intrastromally with isotonic saline as controls. Central corneal thickness and endothelial cell counts were measured before and at 6 hours, 1 day, and 1 week after the injection was given. Corneas were then harvested for transmission electron microscopy.Only the 0.5% group did not significantly recover from corneal edema 1 week after the injection (P = 0.167, P = 0.051, P = 0.086, P = 0.001 in groups A–D, respectively). There were significant differences in endothelial cell counts for the 0.1% and 0.25% groups (P = 0.077, P = 0.019, P = 0.008 in groups A–C, respectively). Transmission electron microscopy evaluation revealed definite necrotic changes in endothelial cells at concentrations of 0.1%, 0.25%, and 0.5%, but only microstructural changes at a concentration of 0.05%.Our results suggest that voriconazole could be injected safely into the intrastromal layer at a concentration of 0.05% with low endothelial cell toxicity. However, injections should be administered with caution because of the risk of microstructural damage.