BACKGROUND: In July 2007, three neonates in the neonatal intensive care unit (NICU) of Chosun University Hospital expired due to Escherichia coli sepsis. An E. coli outbreak was suspected. METHODS: To investigate the outbreak, environmental cultures were taken from NICU. We performed repetitive extragenic palindromic (rep)-PCR to compare genotypes of the three isolates from the cases and one environmental strain of E. coli. A case-control study was done in order to identify risk factors for the infection. RESULTS: In July 2007, the attack rate of E. coli was 11.1%, which was higher than the basal rate. All the three E. coli isolates from the cases presented the same antimicrobial susceptibility pattern whereas other E. coli isolated from non-outbreak period presented different patterns. Among environmental cultures, only one specimen collected from the surface of a bathtub for neonates was culture positive for E. coli. Three strains of the cases and one environmental strain of E. coli showed the same rep-PCR pattern, while control strains showed different patterns. No statistically significant difference in risk factors was found between the case and control groups in the case-control study. CONCLUSION: The result of rep-PCR assay showed that the outbreak had originated from a single clone of E. coli. But we could not identify risk factors for the infection. The attack rate of E. coli in NICU returned to the basal level after implement of the infection control measures such as disinfection of NICU environment and equipments, thorough hand washing, and education of health care workers.