Colorimetric methods using 4-nitrophenyl-glycoside substrates for the assay of beta-galactosidase and N-acetyl-beta-glucosaminidase in human urine are described. Interfering substances were removed by gel-filtration of urine. An unidentified low-molecular-weight inhibitor of N-acetyl-beta-glucosaminidase was found. Increased sensitivity of the methods was achieved by high sample volumes, small volumes of buffer to terminate the enzyme reaction and optimal substrate concentration and buffer pH. Incubation periods were shortened to 15 min. Both methods were designed as single-tube tests in which buffer-substrate solutions are prepared in bulk and aliquots are stored in individual containers at -25 degrees C. Under these conditions reagents were stable for at least six months. Precision of both methods was satisfactory. Estimates of normal limits are reported.