The human kidney is a comparatively rich source of the enzyme acid phosphatase; the highest activity is found in the glomeruli. Since glomerular damage might be reflected by excretion of large quantities of this enzyme into the urine, the present study was undertaken to determine the biochemical characteristics of renal acid phosphatase as it appears in human urine and to establish optimum assay conditions for quantitative measurement of urinary enzyme activity. Enzyme kinetics, including substrate concentration requirement, Km values, and incubationtime characteristics, were determined for renal and urinary acid phosphatase. Stability of enzyme activity during storage and the effect of activators and inhibitors of the enzyme were examined. The influence of acid phosphatase activity derived from sources other than the kidney on the measurement of renal enzyme activity in the urine had to be considered; therefore, the selective inhibitory effects of various compounds on acid phosphatase derived from human kidney, prostate, and red and white blood cells have been studied. Renal and prostatic acid phosphatase share similar biochemical features, particularly with regard to selective inhibition. It was not possible to inhibit the urinary acid phosphatase contributed by prostate without simultaneously strongly affecting renal acid phosphatase. On the other hand, a selective inhibition of the urinary acid phosphatase contributed by erythrocytes and partially that contributed by leukocytes could be obtained. Bacteriuria was considered as a further source of error. The results indicate that it may be possible to utilize the measurement of urinary acid phosphatase activity as an index of glomerular damage in children and adult females but not in adult males, because of the contribution of prostatic acid phosphatase.