A reliable enzyme-linked immunosorbent assay designed to detect N-acetyl-beta-D-glucosaminidase isoenzyme B (NAG-B) was used to determine distribution and variation of urinary NAG-B in normal and pathologic urine. NAG-B values varied over a much broader range in urine from male than in that from female subjects under different conditions of sampling. Because NAG-B values are markedly high in the semen (5800.2 micrograms/liter on the average), contamination of the urine with NAG-B from genital tissues occurs at urination, when it enters urine containing NAG-B of renal origin. The clinical significance of NAG-B and total NAG enzymatic activity as a renal tubular marker should be carefully evaluated when analyzing urine of males from reproductive age.