The rate of occurrence of the extended-spectrum beta-lactamase (ESBL)-producing phenotype among Escherichia coli isolates in Tel Aviv is 12% (22). The aim of this study was to understand the molecular epidemiology of E. coli ESBL producers and to identify the ESBL genes carried by them. We studied 20 single-patient ESBL-producing E. coli clinical isolates. They comprised 11 distinct nonrelated pulsed-field gel electrophoresis (PFGE) genotypes: six isolates belonged to the same PFGE clone, four other clones included two isolates each, and six unrelated clones included only one isolate. All isolates produced various beta-lactamases with pIs ranging from 5.2 to 8.2, varying within similar PFGE clones. The most prevalent ESBL gene was bla CTX-M; 16 isolates carried blaCTX-M-2 and three carried a new ESBL gene designated blaCTX-M-39. Three strains carried WUSHV (two blaSHV-12 and one blaSHV-5), and two strains carried inhibitor-resistant ESBL genes, blaTEM-33 and blaTEM-30; 18 strains carried blaTEM-1 and eight strains carried bla OXA-2. Plasmid mapping and Southern blot analysis with a CTX-M-2 probe demonstrated that blaCTX-M-2 is plasmid borne. The wide dissemination of ESBLs among E. coli isolates in our institution is partly related to clonal spread, but more notably to various plasmid-associated ESBL genes, occurring in multiple clones, wherein the CTX-M gene family appears almost uniformly. We report here a new CTX-M gene, designated bla CTX-M-39, which revealed 99% homology with blaCTX-M-26, with a substitution of arginine for glutamine at position 225.