We assessed the occurrence and screening criterion for extended-spectrum beta-lactamases (ESBLs) among AmpC-producing Enterobacter cloacae, Citrobacter freundii, and Serratia marcescens. The 413 isolates (158 E. cloacae, 126 C. freundii, and 129 S. marcescens) isolated from 11 clinical laboratories in Korea were investigated. ESBL production was confirmed by double-disk synergy test and inhibitor-potentiated diffusion test using ceftazidime (CAZ), cefotaxime (CTX), aztreonam (AZT), and cefepime (FEP) with or without clavulanic acid. One hundred seven isolates (25.9%) were as ESBL producers. Of them, resistance was transferred by conjugation in 82 isolates. In transconjugants, structural genes for CTX-M (53.7%), TEM (46.3%), SHV (29.3%) were found. To evaluate the ESBL screening minimum inhibitory concentration (MIC) criteria, MICs for cefuroxime, CAZ, CTX, AZT, and FEP were determined and cutoff value was selected using receiver operator characteristic curve. The FEP MIC > or = 1 microg/mL had the highest sensitivity (95.3%), specificity (82.7%), and positive (65.8%) and negative predictive values (98.3%).