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Outbreaks of sepsis associated with Acinetobacter baumannii have been rarely reported in neonatal intensive care units (NICUs). We describe such an outbreak in a NICU, and the results of molecular epidemiologic investigations are presented.Between August and September 2000, 6 premature infants hospitalized in a pediatric NICU developed A. baumannii sepsis. Three additional cases had Acinetobacter infections during November and December. For an environmental culture survey, 94 environmental specimens and hand washings of all 43 health care workers involved in this unit were examined for the presence of this organism. Two genotyping methods, pulsed field gel electrophoresis of genomic DNA digested with Sma I and infrequent restriction site polymerase chain reaction, were used to analyze the 9 bacteremic isolates and any A. baumannii isolates obtained from the environmental survey and the hand washings. Another 3 bacteremic isolates of A. baumannii collected in the same NICU in 1999 were incorporated as controls.The 9 infants were premature and had birth weights of <1500 g. Before onset of sepsis 9 infants had received total parenteral nutrition, and 8 infants had had central venous catheters and received intrafat emulsion. Five (5.3%) environmental specimens and 10 (23.3%) hand washing specimens were positive for the organism. Except for the strain from Case 9, the results of both genotyping methods were concordant; 11 patterns were identified by infrequent restriction site polymerase chain reaction and 10 patterns by pulsed field gel electrophoresis. One major genotype was demonstrated in the first 6 bacteremic isolates as well as 3 hand washing isolates. The genotypes of the other 3 bacteremic isolates, the 3 control strains, the 5 environmental isolates and 7 other hand washing isolates were distinct from the genotype of outbreak strains.An outbreak of A. baumannii bacteremia in a NICU was clearly demonstrated by the molecular epidemiologic investigation and was possibly transmitted via the hands of health care workers.