We report a pilot study testing the hypothesis that Gram-negative bacilli colonizing the gastrointestinal tracts of infants with birth weights <1500 g are the source of subsequent bloodstream infections. Ninety-five percent (18 of 19) of paired bloodstream infection or antecedent rectal cultures were genotypically concordant. The gastrointestinal tract is the reservoir for most cases of Gram-negative sepsis in this population.