Urinary excretion of N-acetyl-beta-D-glucosaminidase (NAG) was shown to be reproducible in random urine specimens when expressed as the ratio of NAG to milligrams of urinary creatinine. The enzyme/creatinine ratio in 815 healthy people was relatively constant throughout childhood and adult life except for the first two years after birth and in individuals 56 years or greater. High ratios in the young children may be explained by low urinary creatinine excretion probably related to small body mass and reduced glomerular filtration rate at this age. The ratio was increased in adult uremic patients and children and adults with a variety of neurologic and obstructive lesions of the voiding mechanism. The presence of bacteriuria did not appear to increase the ratio. Significant enzymuria (greater than 2 SD above the mean for age and sex) was detected in 38 of 81 children with well-characterized renal disease. Among patients with predominantly glomerular disorders there was a close relationship between activity of the disease and enzymuria. In patients with tubulointerstitial disease enzymuria was frequent even in the absence of proteinuria. One of the highest enzyme/creatinine ratios was observed in a child with cystinosis. These studies indicate that NAG enzymuria is a sensitive indicator of activity of renal disease and may prove to be a suitable screening test for significant renal disease or injury in childhood.