We describe the first direct testing of the antimicrobial susceptibilities of bacterial pathogens in human clinical fluid samples by the use of ATP bioluminescence. We developed an ATP bioluminescence assay that eliminates somatic sources of ATP to selectively quantify the bacterial load in clinical urine specimens with a sensitivity of <1,000 CFU per milliliter. There was a log-log relationship between light emission and the numbers of CFU in clinical urine specimens. A clinical study was performed to evaluate the accuracy of the ATP bioluminescence assay for determination of the antimicrobial susceptibilities of uropathogens in clinical urine specimens tested in a blinded manner. ATP bioluminescent bacterial density quantitation was used to determine the inoculation volume in growth medium with and without antibiotics. After incubation at 37°C for 120 min, the ATP bioluminescence assay was repeated to evaluate the uropathogen response to antibiotics. The ability of the ATP bioluminescence assay to discriminate between antimicrobial susceptibility and resistance was determined by comparison of the results obtained by the ATP bioluminescence assay with the results obtained by standard clinical microbiology methods. Receiver operator characteristic curves were used to determine the optimal threshold for discriminating between susceptibility and resistance. Susceptibility and resistance were correctly predicted in 87% and 95% of cases, respectively, for an overall unweighted accuracy of 91%, when the results were stratified by antibiotic. For samples in which the pathogen was susceptible, the accuracy improved to 95% when the results for samples with less than a 25-fold increase in the amount of bacterial ATP in the medium without antibiotics were excluded. These data indicate that a rapid bioluminescent antimicrobial susceptibility assay may be useful for the management of urinary tract infections.The urinary tract is the most common organ system to experience bacterial infections (17). As a major cause of patient morbidity and health care expenditures for men and women of all age groups, urinary tract infections (UTIs) account for 7 million office visits, more than 1 million emergency room visits, and 100,000 hospitalizations each year (20). The estimated annual cost to the U.S. health care system is approximately $1.6 billion (8). Patients with nosocomial and/or complicated UTIs due to abnormal urinary tract anatomy, the presence of indwelling foreign bodies, and obstructed kidney stones are at risk for life-threatening systemic infections and permanently reduced kidney function. Patients with recurrent communityacquired, simple UTIs may incur significant treatment costs and substantial morbidity due to bothersome symptoms, such as painful urination, frequency, and urgency.The traditional basis for the evaluation of urinary tract pathogens (uropathogens) is urine culture and antibiotic susceptibility testing (13). The major drawback of the current microbiology approach is the time lapse of 2 to 3 days between s...