The aim of this study was to investigate the presence of extended spectrum beta-lactamase (ESBL), KPC-type carbapenemase and plasmid-mediated AmpC beta-lactamase (pAmpC) which have increased in incidence in recent years in Escherichia coli and Klebsiella pneumoniae strains isolated from the blood samples causing serious infections. Ninety nine E.coli and 114 K.pneumoniae strains which were isolated from the blood samples of patients admitted to Hacettepe University Medical Faculty, Ihsan Dogramaci Children's Hospital between January 2001 and March 2009 were investigated. The screening tests for ESBL, pAmpC beta-lactamase and KPC-type carbapenemase were performed on the same plate. Combined disk test was performed for ESBL and modified Hodge test was done for KPC-type carbapenemase confirmation according to Clinical and Laboratory Standards Institute (CLSI) guidelines. In addition the inhibitory-based test with boronic acid for KPC-type carbapenemase, pAmpC beta-lactamase and the modified Hodge test for pAmpC beta-lactamase were performed. Boronic acid inhibition test was performed to detect the co-presence of the three types of resistance. The frequency of the beta-lactamases in E.coli and K.pneumoniae isolates were as follows respectively: ESBL 26.2% and 61.4%; pAmpC 1% and 0.9% and ESBL + pAmpC 6% and 3.5%. ESBL was masked by pAmpC in an isolate. Ertapenem resistance was shown in three isolates and KPC-type carbapenemases were detected positive by the inhibitory-based test with boronic acid but found to be negative by the modified Hodge test. The results of modified Hodge test was considered valid according to CLSI comments. Since both ESBL and pAmpC were positive but modified Hodge test was negative in these three strains, ertapenem resistance was attributed to another mechanism. For the determination of ESBL and pAmpC beta-lactamases in the routine laboratory, reliable and sensitive susceptibility tests should be performed. The inhibitory-based test with boronic acid is easy for interpretation, and a practical method for the detremination of pAmpC beta lactamases. For KPC-type carbapenemases modified Hodge test which has been standardized by CLSI, is a reliable method. The results of this study showed that ESBL positivity rates are alarming and although the frequency of pAmpC is currently low, it is increasing together with ESBL. These data indicated the need for the establishment of urgent measures to control the increase in plasmid-mediated antibiotic resistance in gram-negative enteric bacteria.