Despite its theoretical advantages, direct antimicrobial susceptibility testing (DST) of urine specimens remains controversial largely because of concerns regarding its accuracy, particularly with mixed cultures. To evaluate the performance of DST in the setting of acute urinary tract infection (UTI), we performed DST using 25 traditional and contemporary antimicrobial agents on urine specimens from 162 women with suspected acute uncomplicated UTI, and compared these results with the results of standardized disk diffusion susceptibility tests done on the same specimens. Direct tests were interpretable for 129 specimens, i.e., 80% of all specimens and 85% of the 152 specimens that met the culture criteria for UTI. Of the 2,983 individual comparisons between the direct and standard tests, 0.8% represented very major errors, 0.6% represented major errors, 3.1% represented minor errors, and 95.5% were in agreement. Errors were more common in association with older antimicrobial agents and agents with a high prevalence of antimicrobial resistance, non-Escherichia coli strains, low urine bacterial concentrations, sparse or mixed growth in the direct test, and the presence of multiple significant organisms in urine. The urine leukocyte concentration was ≥15/mm3 in all subjects and did not differentiate between specimens that gave an interpretable direct test and those that did not. Calculation of the sensitivity of DST in identifying antimicrobial resistance supplemented conventional error rate analysis. We conclude that when used selectively and interpreted carefully, DST of urine specimens offers an efficient, rapid, and accurate method for antimicrobial susceptibility determination for acute UTI, particularly when the urine bacterial concentration is >105 CFU/ml.