Two rapid modifications of a tube assay for the detection of β-glucuronidase activity (PGUA assay) were evaluated for the identification of Escherichia coli from urine cultures. A microwell and filter paper adaptation of the PGUA assay were tested using 1,234 oxidase-negative, Gram-negative rods isolated from urine on MacConkey agar in clinically significant numbers. There was perfect correlation between both methods and 676 of 797 E. coli isolates were PGUA-positive within 2 h while all of remaining isolates were PGUA-negative (sensitivity = 85%; specificity = 100%). We conclude that either modified format of the PGUA assay provides a useful, inexpensive, and rapid alternative spot test for the definitive identification of E. coli from urine because of the high degree of specificity.