The incidence and transmission patterns of extended-spectrum-beta-lactamase (ESBL)-producing Klebsiella pneumoniae in patients admitted to the intensive care unit (ICU) of a university hospital were investigated over a 3-year period. K. pneumoniae isolates were characterized by antibiotic susceptibility, capsular serotyping, plasmid profiles, and pulsed-field gel electrophoresis (PFGE) of genome macrorestriction patterns with XbaI, and the results were compared with those obtained by typing with the randomly amplified polymorphic DNA (RAPD) patterns. The discriminatory power of RAPD typing was evaluated for three primers. The incidence of isolation of ESBL-producing K. pneumoniae was 2.5 cases per 1,000 admissions to the ICU versus 0.35 cases per 1,000 admissions to other units (relative risk, 7.03; 95% confidence interval, 3.89 to 12.69). Infection developed in 53% of evaluable patients. Thirty-six percent of the cases were possibly acquired in other institutions. Isolates from ICU patients were subdivided into six capsular serotypes and into four clonal groups based on antibiotype, plasmid content, and PFGE and RAPD patterns. Two clones were associated with clusters of cross-infection, involving 5 and 12 patients, respectively. Following implementation of contact isolation precautions, the incidence of nosocomial acquisition of ESBL-producing K. pneumoniae decreased from 0.55 to 0.26 cases per 1,000 admissions (P = 0.03). PFGE and RAPD analysis showed concordant results and comparable discrimination for differentiation between groups of epidemiologically related strains of ESBL-producing K. pneumoniae. More subclonal variants were determined among epidemic clones by PFGE analysis than by RAPD analysis. Both methods are useful for typing K. pneumoniae strains in epidemiological investigations, although RAPD analysis is more efficient.