The epidemiology of extended-spectrum beta-lactamase (ESBL)-producing strains of Klebsiella pneumoniae was studied over a 16-month period in a medical intensive care unit (ICU). A control program involving enhanced isolation procedures, surveillance cultures at admission and then at 1-week intervals, and selective digestive decontamination (SDD) was instituted. Phenotypic and genotypic markers (plasmid content and DNA macrorestriction polymorphism determined by pulsed-field gel electrophoresis) were used to compare 138 strains of ESBL-producing K. pneumoniae. The incidence of colonization and/or infection with ESBL producers was 11.9%. ESBL-producing K. pneumoniae strains were isolated from 64 of 65 patients. Fifty-five cases were considered acquired in the ICU, while nine cases were imported. Forty-five infections occurred in 32 patients; 20 infections involved the urinary tract. SDD failed to reduce the incidence of acquisition of ESBL-producing K. pneumoniae. Combined use of markers was necessary to achieve accurate differentiation of strains. A single epidemic clone (SHV-4 beta-lactamase-producing K. pneumoniae) was the cause of 85% of the ICU-acquired cases. Sporadic occurrence of SHV-5, TEM-3, SHV-2, and SHV-3 producers accounted only for a few cases.