BACKGROUND: Escherichia coli and Klebsiella pneumoniae resistant to 3rd generation cephalosporin have been reported with increasing frequency in tertiary-care hospital in Korea. MicroScan Neg Combo Panel Type 21 (Type 21) contains a 1 microgram/mL cepfodoxime (POD) in addition to other screen wells containing ceftazidime, cefotaxime, ceftriaxone, and aztreonam, which are designed for detecting extended-spectrum beta-lactamase (ESBL)-producing E. coli and Klebsiella species. We evaluated the Type 21 panel for its ability to detect ESBL. METHODS: From November to December in 1998, 496 E. coli and 326 K. pneumoniae strains isolated from clinical specimens were tested with Type 21 panel The isolates flagged as ESBL producers by the panel were confirmed by the double disk synergy test (DDS). To evaluate the specificity of POD, n-lactamases of 54 E, coli and 20 K. pneumoniae strains that were flagged by, POD only from January to May 1999 were analyzed by isoelectric focusing(IEF). RESULTS: 75/496(15%) E. coli and 68/326(21%) K. pneumoniae were flagged as ESBL producers by Type 21 panel. Of those, 94 isolates including 38/75 (51%) of E. coli and 56/68 (82%) of K. pneumoniae were positive for DDS. Among the 94 ESBL producers, all were detected by POD, 84% by cefotaxime, 85% by ceftazidime, 84% by ceftriaxone, and 86% by aztreonam. The 74 strains that were flagged as ESBL producers by POD screen well only were mostly DDS-negative, cefoxitin- resistant and showed beta-lactamases with pls of 5.4 and 7.6 or no band, which could be interpreted as the presence of TEM-1 or SHV-1 type beta-lactamases and/or basal AmpC beta-lactamases, not ESBL. CONCLUSION: MicroScan Neg Combo Panel Type 21 was able to detect a greater number of ESBL producers by inclusion of POD in its screening well. However, the specificity of POD was compromised by flagging a significant number of DDS negative strains. We conclude that the isolates with reduced susceptibility to 3rd generation cephalosporins as well as POD can be reported as ESBL-producers and those resistant to POD only should be confirmed by DDS.