In total, 438 (1.7%) Escherichia coli and 125 (3.98%) Klebsiella pneumoniae isolates were found to be producers of extended-spectrum beta-lactamase (ESBL) during 1995-2003 in southern Spain. There was a significant increase in the frequency of ESBL-producing E. coli isolates, from < 0.36% before 1999 to 4.8% in 2003, while the frequency of ESBL-producing K. pneumoniae isolates decreased during the same period. The most common ESBLs detected in K. pneumoniae were SHV type, whereas both CTX-M and SHV types were detected in E. coli. In addition, E. coli isolates showed greater clonal diversity (84 distinct REP-PCR patterns, compared with five in K. pneumoniae), fewer enzymes per isolate, and a higher number of isolates recovered from outpatients. These differences may have implications for the control measures that should be used for these two microorganisms.