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Objectives To assess the frequency and diversity of extended spectrum β-lactamases (ESBLs) in the Champagne-Ardenne region France, and to identify genetic elements associated with the blaCTX-M genes. Methods During 2004, all the non-duplicate isolates of Pseudomonas aeruginosa and Acinetobacter baumannii resistant to ceftazidime and of Enterobacteriaceae intermediate or resistant to ceftazidime and/or cefotaxime, screening samples excluded, were collected in 10 public hospitals and 3 private clinics. bla genes were sequenced and blaCTX-M environment characterized by PCR mapping. Results In Enterobacteriaceae (138/21 861; 0.6%), ESBLs were predominantly TEM-24 (n = 52; 37.7%) and CTX-M-15 (n = 37; 26.8%). Three new enzymes were identified, CTX-M-61 (CTX-M-1 group), TEM- and SHV-type. A. baumannii (n = 5) produced VEB-1 and P. aeruginosa (n = 2) SHV-2a. ISEcp1 was detected in 22/27 strains, disrupted in 7 of them. The IS903-like element was downstream of blaCTX-M-14 and blaCTX-M-16. ISCR1 was found upstream of blaCTX-M-2 and blaCTX-M-9, and ISCR1 and blaCTX-M-2 were located on a sul1-type class 1 integron. In comparison with 2001–02, ESBL distribution among Enterobacteriaceae showed an increase in CTX-M-type (44.9% vs 3.7% P Escherichia coli CTX-M-15 and to the almost total disappearance of TEM-3 (0.9% vs 51.2%). E. coli was the most frequent species (50.0% vs 5.1% in 1998) despite a similar prevalence to that in 1998 (0.5% vs 0.2%). Conclusions A careful detection of blaCTX-M-type spread to other species would help to anticipate clonal endemics such as those observed in Enterobacter aerogenes TEM-24. [ABSTRACT FROM AUTHOR]