A new VITEK 2 antibiotic susceptibility testing (AST) card, AST N-054, was introduced for aerobic gram-negative bacilli in 2007 and has been widely adopted for routine use in the UK. We evaluated its performance for detecting extended-spectrum beta-lactamase (ESBL) production in Escherichia coli. ESBL-producing faecal isolates of E. coli (n = 137) from residents in nursing homes were tested using the AST N-054 card on VITEK 2 and with MASTDISCS ID ESBL detection disc diffusion tests (Mast Diagnostics, Bootle, UK). The susceptibility result recommended by the VITEK 2 software was also recorded. The AST N-054 card detected ESBL production in 93 of the 137 isolates tested [test sensitivity 67.9% (95% CI, 59.7-75.1)]. E. coli strain A, a widespread lineage in the UK with a low-level CTX-M enzyme production, accounted for most of the detection failures, with 35/73 strain A isolates incorrectly reported versus 9/64 non-strain A isolates (P < 0.0001). The MASTDISCS correctly detected ESBL in 135/137 isolates [test sensitivity 98.5% (95% CI, 94.5-99.9)]. Of the 44 isolates found to be negative for ESBL production by VITEK 2, the Advanced Expert System misreported 29 as susceptible to cefotaxime and all as susceptible to ceftazidime and aztreonam. These data suggest that the AST N-054 card for the VITEK 2 system is less reliable than other previously reported cards for the detection of CTX-M beta-lactamase-producing E. coli circulating in the UK, particularly strain A isolates.