Among the 443 clinical isolates of Escherichia coli and Klebsiella spp. collected between June and November 2003 from 3 university hospitals in Korea, 62 isolates were confirmed as extended-spectrum beta-lactamase (ESBL)- or plasmid-mediated AmpC beta-lactamase-producers by double disk synergy test, PCR and sequencing for beta-lactamase genes. The most frequently identified ESBL gene among E. coli and K. pneumoniae isolates was bla(SHV-12) and bla(CTX-M) (bla(CTX-M-9), bla(CTX-M-14), bla(CTX-M-3), and bla(CTX-M-15)). Four kinds of plasmid-mediated AmpC beta-lactamases, ACT-1, CMY-1, CMY-2, and DHA-1, were detected. ESBL production was associated with high levels of resistance to tetracycline, sulfisoxazole, streptomycin, kanamycin, gentamicin and tobramycin when compared to non-ESBL producing isolates. Conclusively, this study suggests that the CTX-M beta-lactamases are prevalent and various kinds of plasmid-mediated AmpC enzymes are distributed in clinical isolates of E. coli and Klebsiella spp. in Korea.