A chromogenic Limulus amoebocyte lysate assay, performed at room temperature, was evaluated for rapid detection (five minutes) of asymptomatic bacteriuria of pregnancy. Development of a distinctive yellow color was used to detect urines containing greater than 10(5) gram-negative bacteria per milliliter. One thousand thirty-nine urine samples were obtained from 664 obstetric patients, with 86 of the specimens shown to contain greater than 10(5) gram-negative urinary tract pathogens. Of these, 74 of 86 were detected by chromogenic Limulus assay; 11 false-positive tests were recorded. At a urine dilution of 1:10, sensitivity and specificity were 88.7 and 98.7%, respectively. Predictive values, based on an attack rate of 10%, were 98.6% for a negative test and 89.9% for a positive test. These data suggest that chromogenic Limulus amoebocyte lysate assay of urine has potential usefulness as a rapid, reliable, and easily performed and interpreted screening test for asymptomatic bacteriuria of pregnancy.