We describe an outbreak of nosocomial respiratory infection caused by multi-drug resistant Acinetobacter baumannii in a neonatal intensive care unit (NICU) in Tunis and our investigation to determine the source. Between May 2006 and February 2007, 31 infants hospitalized in the NICU of the Centre of Maternity and Neonatology of La Rabta in Tunis developed A. baumannii pneumonia. A case (infected infant) was defined as any patient hospitalized in the NICU during the outbreak period, with clinical signs of pneumonia and isolation of A. baumannii from tracheal aspirate. Ten rectal swabs and 98 environmental specimens were collected for the epidemiological investigation. Thirty-nine A. baumannii isolates were collected: 31 clinical strains from tracheal aspirates (>10(3) colony-forming units [CFU]/mL), 3 environmental strains from incubators, and 5 from rectal swab. For the genotyping method, we used pulsed-field gel electrophoresis using ApaI restriction endonuclease. Thirty-one neonates developed multiple drug-resistant A. baumannii-associated pneumonia with 10 deaths due to A. baumannii infection, 48.4% had very low birth weight (<or=1500 g), and 67.7% neonates were premature. The mean age at the beginning of symptoms was 6.9 days. A. baumannii isolates were resistant to all beta-lactams. Resistance rates to other antibiotics were, respectively, 94.9% for gentamicin, 87.2% for cotrimoxazole, 41% for netilmicin, and 5.1% for tobramycin. All the isolates were susceptible to colistin. Pulsed-field gel electrophoresis analysis of outbreak-isolates indicated the presence of only one clone (A) containing nine subtypes genetically related to the outbreak strain. The clonal diffusion of A. baumannii strains in an NICU was confirmed by molecular method. Control measures were reinforced to contain the outbreak.