The correlation between 24-hour urinary excretion of N-acetyl-β-d-glucosaminidase (NAG) and γ-glutamyl transferase (GGT) with urine NAG and GGT/creatinine ratios was assessed in dogs with gentamicin-induced nephrotoxicosis. Eighteen 6-month-oid male Beagles with normal renal function were randomly divided into 3 groups of 6. Each group was fed a different concentration of protein (high protein, 27.3%; medium protein, 13.7%; and low protein, 9.4%) for 21 days. After dietary conditioning, gentamicin was administered at a dose of 10 mg/kg IM tid for 8 days and each group was continued on its respective diet. Endogenous creatinine clearance and 24-hour urinary excretion of NAG and GGT were determined after dietary conditioning (day 0) and on days 2, 4, 6, and 8 of gentamicin administration. In addition, urine NAG and GGT/creatinine ratios (IU/L ± mg/dL) were determined from catheterized spot urine samples obtained between 7 and 10 am on the same days. The correlation between 24-hour urinary enzyme excretion and urine enzyme/creatinine ratio in the spot urine samples was evaluated by simple linear regression analysis. Spot sample urine enzyme/creatinine ratios were significantly correlated with 24-hour urinary enzyme excretion through day 4 for dogs on low dietary protein, through day 6 for those on medium protein, and through day 8 for those on high dietary protein. Mean ± SD baseline values for urine NAG/creatinine ratio and 24-hour urinary NAG excretion were 0.06 ± 0.04 and 0.19 ± 0.14 IU/kg/24 hr, respectively. Baseline values for urine GGT/creatinine ratio and 24-hour urinary GGT excretion were 0.39 ± 0.18 and 1.42 ± 0.82 IU/kg/24 hr, respectively.