Necrotizing enterocolitis (NEC) is the most common gastrointestinal emergency in the neonatal period. Small-bowel overgrowth with aerobic gram-negative bacteria has previously been implicated in the development of NEC. This prospective study performed quantitative bacteriology on 422 duodenal aspirates collected from 122 very-low-birth-weight (<1,500-g) newborns, at the time of routine changing of nasogastric tubes. Isolates of Enterobacteriaceae were typed by repetitive extragenic, palindromic PCR and pulsed-field gel electrophoresis. One or more samples from 50% of these infants yielded gram-negative bacteria, predominantly Escherichia coli, Klebsiella spp., and Enterobacter spp., with counts up to 10⁸ CFU/g. The proportion of samples with gram-negative bacteria increased with postnatal age, while the percentage of sterile samples declined. Molecular typing revealed marked temporal clustering of indistinguishable strains. All infants had been fed prior to isolation of gram-negative organisms. Antibiotic use had no obvious effect on colonization with Enterobacteriaceae. There were 15 episodes of suspected NEC (stage I) and 8 confirmed cases of NEC (2 stage II and 6 stage III) during the study period. Duodenal aspirates were collected prior to clinical onset in 13 episodes of NEC. Seven of these yielded Enterobacteriaceae, of which five strains were also isolated from infants without NEC. Very-low-birth-weight infants have high levels of duodenal colonization with Enterobacteriaceae, with evidence of considerable cross-colonization with indistinguishable strains. There was no association between duodenal colonization with particular strains of Enterobacteriaceae and development of NEC.