An outbreak of extended-spectrum beta-lactamase (ESBL) producing Klebsiella pneumoniae (ESBL-Kp) in a neonatal intensive care unit prompted a prospective surveillance study between 1211 September and 611 October 2003. Surveillance was carried out by obtaining stool samples twice a week. The DNA relatedness of the isolates was shown by random amplified polymorphic DNA comparison (ERIC-PCR). ESBL production was identified by clavulanate synergy, isoelectric focusing, PCR and sequence analysis. During the study period, 49 neonates were hospitalized in the neonatal intensive care unit (NICU). In the first 20-day period, five neonates were infected with ESBLKp. The first patient treated with third generation cephalosporin and the second patient treated with meropenem died. While all three infected survivors were clinically improving, the digestive tracts were being colonized by SHV5 producing Klebsiella.