Multiresistant Klebsiella pneumoniae strains with plasmid-borne extended-spectrum beta-lactamases (ESBL) are increasingly frequent nosocomial pathogens. A major outbreak of clinical infections, mainly involving patients in the Newborn Services Unit with limited spread to adult patients, occurred at our hospital. This epidemic was investigated by typing the isolates phenotypically and with random amplified polymorphic DNA analysis (RAPD) and plasmid analysis. Forty-eight isolates, consisting of 44 consecutive clinical isolates and 4 selected surveillance isolates, were studied. A single decamer primer was used for the RAPD, and this was effective in demonstrating that the majority of isolates (45 of 48) had the same profile. Three other isolates had different RAPD patterns identifying them as nonepidemic strains. Plasmids were extracted by alkaline lysis with Magic-miniprep kits from 10 isolates selected to represent the epidemic and nonepidemic strains. This method produced small (< 20-kb) plasmids; larger ESBL-carrying plasmids were not produced, but the small plasmids nonetheless allowed strain differentiation. Antibiotic susceptibility patterns alone were not reliable as strain indicators, since some isolates with the RAPD pattern characteristic of the epidemic strains did not express ESBL and therefore were susceptible to extended-spectrum cephalosporins. The investigation showed the predominance of a single epidemic strain that was transmitted between patients in the Newborn Services Unit. RAPD was the best of the methods used for detecting strain differences, and its speed and ability to type a wide variety of species suggest that it will be an increasingly useful molecular epidemiologic tool.