NCCLS screening and confirmation methods for detecting extended-spectrum -lactamases (ESBLs) apply only to Escherichia coli and Klebsiella spp., yet ESBLs have been found in other members of the family Enterobacteriaceae. We evaluated the effectiveness of NCCLS methods for detecting ESBLs in 690 gramnegative isolates of Enterobacteriaceae that excluded E. coli, Klebsiella pneumoniae, and Klebsiella oxytoca. Isolates were collected between January 1996 and June 1999 from 53 U.S. hospitals participating in Project ICARE (Intensive Care Antimicrobial Resistance Epidemiology). The antimicrobial susceptibility patterns of the isolates were determined by using the NCCLS broth microdilution method (BMD), and those isolates for which the MIC of ceftazidime, cefotaxime, ceftriaxone, or aztreonam was >2 g/ml or the MIC of cefpodoxime was >8 g/ml (positive ESBL screen test) were further tested for a clavulanic acid (CA) effect by BMD and the disk diffusion method (confirmation tests). Although 355 (51.4%) of the isolates were ESBL screen test positive, only 15 (2.2%) showed a CA effect. Since 3 of the 15 isolates were already highly resistant to the five NCCLS indicator drugs, ESBL detection would have an impact on the reporting of only 1.7% of the isolates in the study. Only 6 of the 15 isolates that showed a CA effect contained a bla TEM , bla SHV , bla CTX-M , or bla OXA -lactamase gene as determined by PCR (with a corresponding isoelectric focusing pattern). Extension of the NCCLS guidelines for ESBL detection to Enterobacteriaceae other than E. coli and Klebsiella spp. does not appear to be warranted in the United States at present, since the test has poor specificity for this population and would result in changes in categorical interpretations for only 1.7% of Enterobacteriaceae tested.First described in 1983 (10), extended-spectrum -lactamases (ESBLs) have contributed to the dramatic increase in resistance to -lactam agents among gram-negative bacteria in recent years (2,3,8). These enzymes, encoded by genes that are typically plasmid borne, hydrolyze penicillins, cephalosporins, and aztreonam and are inhibited by clavulanic acid (CA) (2, 3, 5). Although most commonly produced by Escherichia coli and Klebsiella spp., ESBLs may be harbored by many other gram-negative bacilli as well, including but not limited to many other bacteria in the family Enterobacteriaceae (7,8,25).While most of the currently known ESBLs are derived from the older -lactamases TEM-1, TEM-2, and SHV-1 (2, 11, 25), CTX-M enzymes are also inhibited by CA and are in the category of ESBLs. In addition, some of the enzymes of the OXA family, although belonging to functional group 2d (5), show a CA effect and are considered ESBLs (2).While a variety of phenotypic and molecular methods have been successfully employed to confirm the presence of ESBLs in clinical isolates, their use is too labor-intensive to be practical for routine screening in microbiology laboratories. Since routine susceptibility testing does not always detect the presence of ...