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To investigate age-related effects on human corneal γ-glutamyltranspeptidase (GGT) (ectoenzyme important to maintaining corneal hydration and antioxidant potential via glutathione recapture).Age-related differences between total, endothelial, and epithelial GGT activity and endothelial cell density were determined for corneas from 29 donors (mean age, 53 ± 17 years; age range, 13–83 years). GGT activity was determined using a standard colorimetric assay based on the transpeptidation reaction. Corneal GGT localization and expression was determined by immunohistochemistry.Total corneal, endothelial, and epithelial GGT activities in the young (<50 years) donor corneas were 37% (P = 0.02), 44% (P = 0.001), and 36% (P = 0.06) higher, respectively, than in the senior (≥50 years) corneas. The age-related rates of decline for GGT activity were 1.0 unit per year for total cornea, 0.4 to 0.5 unit per year for endothelium, and 0.3 to 0.4 unit per year for epithelium. Notably, endothelial cell density in the young corneas was 14% (P = 0.001) higher than in the senior corneas declining about 100 cells per square millimeter per decade (0.3% per year). GGT activity per 106 endothelial cells decreased at about 0.2 units per year and GGT activity per 106 endothelial cells in the young corneas was 41% higher (P = 0.01) than in the senior corneas. Fewer immunoreactive GGT-positive epithelial cells were detected in senior cornea.The age-related loss of human corneal GGT activity was associated with reductions in endothelial and epithelial GGT activity, being because of reduced number of GGT-positive endothelial and epithelial cells and reduced GGT activity per endothelial cell.