Fourteen renal biomarkers were compared with measurement of glomerular filtration rate (GFR) in detecting acute kidney injury (AKI) in beagle dogs given gentamicin (40 mg/kg/day by subcutaneous injection) for 7 consecutive days. Serum and urinary biomarkers were measured before administration of gentamicin and then on days 4 and 8 after starting administration. GFR was derived by use of a simplified equation. Increased urinary cystatin C and decreased GFR occurred from day 4 and were detected before increases in blood urea nitrogen (BUN) and serum creatinine concentrations and changes in other urinary parameters. The closest correlation was between urinary cystatin C and GFR. At termination, microscopical examination revealed extensive necrosis of proximal tubular epithelium with hyaline casts in the kidney of treated dogs. These data indicate that urinary cystatin C is the most sensitive index of kidney injury and GFR reflects the kidney functional mass.