The aim of this study was to identify the types of extended-spectrum beta-lactamases (ESBL) produced by 12 Escherichia coli and 32 Klebsiella spp. (28 K. pneumoniae, 4 K. oxytoca) strains isolated from various specimens (urine, blood, tracheal aspirate, abscess, throat, drain/catheter tips, pleural/peritoneal fluids, cerebrospinal fluid, eye) of patients hospitalized in different units (intensive care, hematology, oncology neonatology, transplantation, pediatric surgery) of Istanbul Medical Faculty Hospital, Turkey. Antimicrobial susceptibility tests were performed by disc diffusion according to NCCLS (CLSI) guidelines and no resistance to imipenem or meropenem was detected. MICs of cefotaxime and ceftazidime were determined by agar dilution method and MIC50 and MIC90 for cefotaxime were found as 16 microg/ml and 64 microg/ml in both Klebsiella spp. and E. coli strains, respectively. The presence of ESBL was confirmed by double-disc synergy testing and E-test ESBL. All isolates demonstrated an ESBL phenotype by these two methods. Isoelectric focusing (IEF) method demonstrated that the isolates produced 1-4 different beta-lactamases (pls: 5.4-9.0). The rates of TEM, SHV, CTX-M beta-lactamases detected by using specific primers in polymerase chain reaction (PCR), were found as 64.3%, 92.9%, 64.3% for K. pneumoniae and 66.7%, 25%, 83.3%, for E.coli strains, respectively. The profiles generated by randomly amplified polymorphic DNA (RAPD)-PCR using ERIC-2 primer revealed several bands, ranging in size from 170 to 1500 bp. According to RAPD-PCR results, K. pneumoniae, K. oxytoca and E. coli strains were separated to 10, 3 and 6 groups, respectively. In the conjugation experiments, 31 of the isolates (70.4%) transferred their resistance genes to recipient E. coli strain. Plasmid analysis studies showed that resistance genes were carried on a single plasmid (> 48 kb) in 20 transconjugants (64.5%), while the rest of the strains (35.5%) harbored more than one plasmid, with sizes ranging from 10 to 100 kb. These results showed the rapid emergence and high prevalence of CTX-M type enzymes among Klebsiella spp. and E. coli strains in our hospital.