Over the past 20 yr, increased attention has been directed toward evaluation of urinary enzymes as markers of nephrotoxicity in dogs because the technique is noninvasive and considered to be more sensitive than the more commonly used conventional tests of renal function. Urinary enzymes also have the potential of determining the primary site of renal damage because different sections of the nephron have a characteristic complement of enzymes. In dogs, increases in brush border enzymes, including gamma-glutamyl transferase and alkaline phosphatase, have been associated with renal proximal tubular damage, while increases in N-acetyl-beta-D-glucosaminidase have been observed in the early stage of renal papillary necrosis. Urinary enzymes have been particularly useful in detection of acute renal damage in dogs, specifically tubular damage: however, their corresponding value in providing information about chronic renal damage remains to be established. Although elevation of certain enzymes appears to be a relatively sensitive measure of nephrotoxicity in the dog, there is no current agreement regarding which enzyme assays are the most appropriate for routine use in safety assessment studies. In addition, elevation of a single enzyme is of limited diagnostic value in detection of renal damage because spurious increases in urinary enzymes sometimes occur in normal dogs. Therefore, if one wishes to conduct special assessment of nephrotoxicity in dogs, evaluation of several enzymes at multiple time points is needed to compensate for normal enzyme variation and to identify potential anatomic site selectivity of the toxin.