The aim of the study is to investigate the prevalence of plasmid-mediated AmpC beta-lactamases in Enterobacteriaceae naturally lacking chromosomal AmpC beta-lactamases. A total of 1860 clinical isolates of Klebsiella spp., Salmonella spp., and Proteus mirabilis were collected from a Korean hospital between January 2002 and December 2004. For the isolates that are nonsusceptible to cefoxitin, polymerase chain reaction amplification of the bla(SHV), bla(TEM), and bla(AmpC) genes and sequencing were performed. Plasmid-mediated AmpC beta-lactamases were found in 2.9% (37 isolates of DHA-1, 1 isolate of CMY-1, 1 isolate of CMY-2, and 1 isolate of ACT-1) of Klebsiella pneumoniae, 2.5% (5 isolates of DHA-1) of Klebsiella oxytoca, 0.8% (1 isolate of DHA-1) of Salmonella spp., and none of P. mirabilis isolates. The DHA-1-producing K. pneumoniae was only 2 isolates (0.6%) in 2002, but the rate and the number significantly increased to 2.4% (13 of 538 isolates) in 2003 and to 4.3% (22 of 512) in 2004. In conclusion, DHA-1 is the most prevalent plasmid-mediated AmpC beta-lactamase in Enterobacteriaceae lacking chromosomal ampC gene, and the DHA-1-producing K. pneumoniae isolates have rapidly increased since 2003 in a Korean hospital. In addition, this is the first report of the appearance of a K. pneumoniae isolate producing ACT-1 beta-lactamase in Korea.