AmpC beta-lactamases are not inhibited by clavulanic acid and could potentially mask detection of extended-spectrum beta-lactamases (ESBLs) using the Clinical and Laboratory Standards Institute confirmatory test. Syn2190 (1,5-dihydroxy-4-pyridone monobactam) selectively inhibits AmpC, but not ESBLs. Fifty-four MicroScan ESBL screen-positive strains of Escherichia coli and an unrelated group of 20 cefoxitin-nonsusceptible E. coli strains were tested with the confirmatory ceftazidime-cefotaxime-clavulanate disk method with or without 4 microg/mL of Syn2190 in the agar. Without Syn2190, 8 (14.8%) of 54 E. coli isolates and 0 of 20 cefoxitin-nonsusceptible E. coli isolates were confirmed. With Syn2190, an additional 9 (16.6%) of 54 of the MicroScan screen-positive E. coli isolates and 6 (30%) of 20 of the cefoxitin-nonsusceptible E. coli isolates were found. Multiplex polymerase chain reaction and sequence analysis confirmed the presence of the plasmid-associated beta-lactamase gene bla(CMY-2) in the 2 available MicroScan-screened E. coli isolates and in 5 of 6 of the cefoxitin-resistant group. These data suggest that in the presence of AmpC, ESBLs in E. coli may not be detected by the currently recommended confirmatory test.