To determine the risk factors for rectal colonization by extended-spectrum beta-lactamase (ESBL) Klebsiella sp. in 368 newborns admitted consecutively to a neonatal intensive care unit over six months, rectal swabs were cultured on admission and weekly until discharge. Eighty infants (21.7%) had ESBL Klebsiella sp. cultured from their rectal swabs. Eighty controls were selected at random from infants with negative cultures admitted within the 14-day period prior to the detection of ESBL Klebsiella sp. in the cases. Cases had significantly lower birth weight, gestational age, earlier age of admission, longer hospital stay, and higher proportions of congenital malformations, early-onset pneumonia and respiratory distress syndrome compared with controls. Significantly more cases received mechanical ventilation, nasal continuous positive airway pressure support, total parenteral nutrition, umbilical vascular catheterization, arterial line insertion, urinary bladder catheterization, and prior treatment with antibiotics. However, stepwise logistic regression analysis showed that only two independent risk factors were significantly associated with ESBL rectal colonization: duration of hospital stay [adjusted odds ratio (OR): 1.3; 95% confidence intervals (CI): 1.2, 1.4; P<0.0001) and early-onset pneumonia (adjusted OR: 8.3; 95% CI: 1.6, 43.4; P=0.01).