Prevalence and characteristics of extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae in Korean hospitals were assessed. A total of 1484 clinical Enterobacteriaceae isolates were collected from 8 tertiary-care hospitals in various regions of Korea over a 3-month period (June to August) in 2005. Among 546 Klebsiella pneumoniae isolates, 123 isolates (22.4%) showed ESBL-producing activity, and 47 (10.2%) of 460 isolates of Escherichia coli were ESBL producers. Of the Enterobacter cloacae isolates, 16.2% (17/105) evidenced ESBL-producing activity. The most prevalent ESBLs were SHV-12 and CTX-M-14 in K. pneumoniae and E. coli, respectively. In E. cloacae, SHV-12 was also the most prevalent. Prevalence of ESBL production differed among the specimens. Although the K. pneumoniae isolates from urine and aspirates evidenced high ESBL production rates (35.4% and 57.1%, respectively), those from sputum, blood, and pus showed relatively low ESBL production rates (17.0%, 14.8%, and 5.3%, respectively). However, E. coli isolates obtained from sputum showed significantly higher ESBL production rates (37.5%) than were seen in samples obtained from other sources, but those obtained from urine showed lower ESBL production rates (8.3%). These significant differences in ESBL-producing K. pneumoniae and E. coli isolates among the isolated specimens should be examined further, with an eye toward the implications of this research in clinical settings.