The aim of this study was to investigate the relationship between the urine excretion and kidney activities of enzymes predominantly located in the proximal renal tubule, viz. the lysosomal hydrolase N-acetyl-β-D-glucosaminidase (NAG) and the predominantly brush border enzymes alanine aminopeptidase (AAP) andγ-glutamyltransferase (GGT) in an experimental model of gentamicin nephrotoxicity. Groups of six animals received either gentamicin (50 mg/kg/day by intraperitoneal injection) or saline daily and were killed after 4, 7, 10, or 14 days of treatment. Gentamicin nephrotoxicity was characterized by reduced creatinine clearance rates and increased urinary flow rate and glycosuria, but only on day 14. Structural changes included a similar degree of vacuolation of the renal proximal convoluted tubules (PCT) in all animals sacrificed on days 11 and 14, some evidence of PCT brush border loss, and the presence of protein casts on day 14. Following gentamicin treatment, increased NAG, AAP, and GGT enzymuria were noted at all time points tested. However, while the increases in urine AAP and GGT activity were paralleled by decreased total renal activity, total kidney NAG activity increased on days 4, 7, and 11 before falling back to pretreatment values on day 14. Interestingly, NAG enzymuria was highest in those animals with protein casts in the lumen of the PCT. The results suggest that increased AAP and GGT enzymuria reflect loss of brush border integrity while increased NAG enzymuria is consistent with the autophagic response of the kidney to acute injury.