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Purpose: To compare the physical and biological characteristics of commercial gamma-irradiated corneas with those of fresh human corneas and to determine suitability for transplantation. Methods: The physical properties of gamma-irradiated and fresh corneas were evaluated with respect to light transmittance, hydration (swelling ratio), elastic modulus (compressive modulus by the indentation method), matrix organization (differential scanning calorimetry), and morphology (light and transmission electron microscopy). The biological properties of the gamma-irradiated cornea, including residual cell content and cellular biocompatibility, were evaluated by quantifying DNA content and measuring the proliferation rate of human corneal epithelial cells, respectively. Results: The hydration, light transmittance, elastic modulus, and proliferation rate of human corneal epithelial cells were not significantly different between fresh and gamma-irradiated corneas. However, differences were observed in tissue morphology, DNA content, and thermal properties. The density of collagen fibrils of the gamma-irradiated corneal sample (160.6 6 33.2 fibrils/mm 2 ) was significantly lower than that of the fresh corneal sample (310.0 6 44.7 fibrils/mm 2 ). Additionally, in the gamma-irradiated corneas, cell fragments—but not viable cells—were observed, supported by lower DNA content of the gamma-irradiated cornea (1.0 6 0.1 mg/mg) than in fresh corneas (1.9 mg/mg). Moreover, the denaturation temperature of gamma-irradiated corneas (61.8 6 1.1 °C) was significantly lower than that of fresh corneas (66.1 6 1.9 °C). Conclusions: Despite structural changes due to irradiation, the physical and biological properties of the gamma-irradiated cornea remain similar to the fresh cornea. These factors, combined with a decreased risk of rejection and longer shelf life, make the gammairradiated tissue a viable and clinically desired option in various ophthalmic procedures.