We tested the hypothesis that enhanced tubular chymase protein expression in diabetic kidneys activates the epithelial sodium channel (ENaC). Diabetic (db/db) and control (db/m) mice were studied during prediabetes, type II diabetes, and progression to type I diabetes at 5-, 18-, and 36-wks of age, respectively. In 5-wk-old diabetic (n=11), body weights (25.6 ± 0.7 vs 20.3 ± 0.6 g) were significantly increased, while plasma glucose (143 ± 29 vs 160 ± 13 mg/dL) and insulin (1.2 ± 0.1 vs 1.2 ± 0.1 μg/L) levels were not different than control (n=11) mice. In 18-wk-old diabetic (n=8) mice, body weights (53.7 ± 0.7 vs 29.7 ± 0.3 g), blood glucose (400 ± 16 vs 137 ± 6 mg/dL), and insulin (20 ± 6 vs 1.9 ± 0.7 μg/L) levels were increased compared to control (n=9) mice. In 36-wk-old diabetic (n=7), body weights (33 ± 3 vs 40 ± 2 g) were decreased, plasma glucose levels (552 ± 40 vs 220 ± 21 mg/dL) increased, and insulin levels (0.8 ± 0.1 vs 0.4 ± 0.1 μg/L) not different from control (n=8) mice. Immunohistochemistry revealed chymase localization to apical membranes of principal cells of connecting tubules and collecting ducts. Densitometric analyses revealed that diabetic renal papillary chymase immunostaining levels were 4 to 28 fold higher relative to control papilla at all ages (p≤0.05). In 5-wk-old diabetic mice, renal cortical chymase expression was increased 1.8 ± 0.3 fold, but decreased to 0.5 ± 0.1 fold in 36-wk-old diabetic compared to control mice (p≤0.05). Relative amiloride-sensitive Na+ currents were increased by chymase (5 μg/mL) perfused Xenopus laevis oocytes injected with α-, β-, and γ- ENaC (1.57 ± 0.08; n=6) relative to mock control (1.13 ± 0.02; n=3) oocytes (p≤0.01). In conclusion, these are the first studies to demonstrate chymase protein localization to the ENaC expressing principal cells of the distal nephron. Papillary chymase expression is unique to prediabetic and diabetic kidneys and does not correlate with glycemia, insulinemia, or body weight. Elevated renal chymase expression may serve as an early biomarker for prediabetic patients with a high risk of developing diabetic renal disease. Enhanced chymase activity, associated with diabetes mellitus, may increase ENaC activity, potentially leading to hypertension and diabetic renal disease.