Studies were carried out on premature infants in the neonatal intensive care unit to determine the effect of feeding of lactobacilli on colonization of the gastrointestinal tract by antibiotic-resistant gram-negative enteric organisms. Thirty premature infants were matched by birth weight and gestational age, randomized and fed double blind either lactobacilli-containing formula or non-lactobacilli-containing formula within 72 hours of delivery. The two study groups were screened weekly by culture for stool lactobacilli, for gram-negative bacteria and for antibiotic resistance of these bacteria. Lactobacilli were cultured from the stools of 13 of 15 patients receiving lactobacilli and from 3 of 15 patients not receiving lactobacilli (P less than 0.001). Gram-negative enteric organisms were isolated during 40 of the 86 weeks (47%) of hospitalization for patients receiving lactobacilli and during 28 of 57 weeks (49%) for patients not receiving lactobacilli. There was no significant difference between the study groups in the number of resistant organisms or in the proportion of resistant organisms per gram-negative enteric isolates (4 of 40 vs. 0 of 28). These results suggest that facultative gram-negative enteric bacterial colonization, with either total or aminoglycoside-resistant strains, is not decreased by oral feedings of Lactobacillus acidophilus in premature infants.