Abstract This study was to determine the prevalence and characteristics of CTX-M–type extended-spectrum β-lactamases (ESBLs) in nonduplicate Escherichia coli ( n = 760) and Klebsiella pneumoniae ( n = 379) bloodstream isolates collected during January 2005 to October 2007 at a university hospital (2000 beds) in Seoul, Korea. Antimicrobial susceptibilities were determined by disk diffusion and agar dilution methods. The double-disk synergy test detected ESBLs in 8.7% (66/760) of E. coli and 11.3% (43/379) of K. pneumoniae isolates. Polymerase chain reaction detected bla CTX-M in 60/66 (90.9%) E. coli and 9/43 (20.9%) K. pneumoniae isolates with the ESBL phenotype. CTX-M-14 was the most common type of CTX-M ESBLs in both E. coli ( n = 32) and K. pneumoniae ( n = 6). CTX-M-15 was the 2nd most common type of CTX-M ESBLs in E. coli ( n = 22), but it was not detected in K. pneumoniae. In addition, CTX-M-24 ( n = 2), CTX-M-65 ( n = 2), CTX-M-27 ( n = 1), and CTX-M-32 ( n = 1) were detected for the 1st time in Korea.