An enzyme capture assay (ECA) for rapid identification of Escherichia coli in blood cultures by using beta-D-glucuronidase as a marker was developed. Microdilution plates coated with antiglucuronidase were used to capture this enzyme from the cell lysates of blood cultures which showed growth of gram-negative bacteria. The assay, using 4-methylumbelliferyl-beta-D-glucuronide as a fluorogenic substrate, had a detection limit of 0.1 ng/ml (3 x 10(-13) M) for the enzyme; this was approximately equal to a cell concentration of 10(6) CFU of E. coli per ml. Among 212 blood cultures showing growth of gram-negative bacteria, 77 specimens were found to contain E. coli by conventional culture procedures and 73 samples were positive by ECA. Among the 135 blood cultures from which E. coli was not isolated, ECA gave one false-positive (Salmonella enteritidis) reaction. Thus, the sensitivity and specificity for the identification of E. coli in blood cultures by ECA were 94.8% (73/77) and 99.3% (134/135), respectively. From the finding of positive growth in the culture bottle, the assay can be completed within 4 h. In view of the high rate of isolation of E. coli from bacteremic patients, the test can be performed in parallel with conventional culture protocols; this may shorten the identification time for E. coli, and proper antimicrobial treatments may be started 24 h earlier than when results of conventional identification systems are used.