tObjective: The present study was designed to evaluate the molecular epidemiology of CTX-Mproducing Klebsiella pneumoniae, Enterobacter cloacae and Escherichia coli isolated from blood-stream infections at tertiary care hospitals in the State of Rio de Janeiro, Brazil.Material and methods: A total of 231 nonduplicate Enterobacteriaceae were isolated fromfive Brazilian hospitals between September 2007 and September 2008. The antimicrobialsusceptibility testing was performed by disk diffusion method according to the Clinical Lab-oratory Standard Institute. Isolates showing resistance to third-generation cephalosporinswere screened for ESBL activity by the double-disk synergy test. The presence of blaCTX-M,blaCTX-M-15and blaKPCgenes was determined by Polymerase Chain Reaction (PCR) amplifica-tion and DNA sequencing. The molecular typing of CTX-M producing isolates was performedby pulsed-field gel electrophoresis (PFGE).Results and discussion: Ninety-three isolates were screened as ESBL positive and 85 (91%)were found to carry CTX-M-type, as follows: K. pneumoniae 59 (49%), E. cloacae 15 (42%), andE. coli 11 (15%). Ten isolates resistant for carbapenems in K. pneumoniae were blaKPC-2genepositive. Among CTX-M type isolates, CTX-M-15 was predominant in more than 50% ofisolates for K. pneumoniae, E. coli, and E. cloacae. PFGE analysis of CTX-M producing isolatesshowed the predominance of CTX-M-15 in 10 of 24 pulsotypes in K. pneumoniae, 6 of 13 in E.cloacae and 3 of 6 in E. coli. CTX-M-15 was also predominant among KPC producing isolates. In conclusion, this study showed that CTX-M-15 was circulating in Rio de Janeiro state in 2007–2008. This data reinforce the need for continuing surveillance because this scenario may have changed over the years.