BACKGROUND: Clinical isolates of AmpC beta-lactamase- producing Enterobacteriaceae were evaluated to determine the prevalence of CTX-M extended-spectrum beta-lactamases (ESBLs) and their genetic environments. METHODS: A total of 250 non-duplicate isolates of Eneterobacter aerogenes, E. cloacae, Citrobacter freundii, Serratia marcescens and Morganella morganii were collected at a Korean hospital. ESBL production was determined by double disk synergy test. For ESBL producers, bla genes were sequenced and blaCTX-M environment was characterized by PCR mapping and sequencing. RESULTS: Among the 250 isolates 29 (11.6%) produced ESBL, and 14 of the 29 isolates produced CTX-M ESBLs, including CTX-M-9 by 8 isolates, CTX-M-3 by 4 isolates, CTX-M-12 by 1 isolate, and CTX-M-14 by 1 isolate. ISEcp1 was present upstream of blaCTX-M-3, 12, and 14. Three of the four CTX- M-3 producers had the same genetic environment (pemK-ISEcp1-blaCTX-M-3-orf477-mucA). An IS903-like element was found downstream of blaCTX-M-14. ISCR1 was identified upstream of blaCTX-M-9 and ISCR1 and blaCTX-M-9 were located on sul1-type class 1 integron. The variable region between the 5'-CS and the first 3'-CS contained dfrA16 and aadA2. Its structure was similar to that of In60, but our isolates did not have IS3000 or second 3'-CS. CONCLUSION: CXT-M type ESBL was prevalent in AmpC beta-lactamase-producing Enterobacteriaceae, particularly E. cloacae. blaCTX-M genes were associated with ISEcp1 or ISCR1. This is the first report on the genetic environment of blaCTX-M in Korean isolates.