Background/Purpose: The authors hypothesized that gastric acidity is protective because it is bactericidal. They tested acidified formula for protection against gut colonization and bacterial translocation. Methods: In vitro: Formula was acidified to pH of 2, 3, 4, 5 and innoculated with Enterobacter. Growth over time was quantitatively assessed. In vivo: 442 premature rabbit pups were sorted randomly and fed formula of pH 2, 3, 4, or 7, with ranitidine. Two models were utilized: (I)with bacterial challenge using a known acid sensitive organism, (2) without bacterial challenge to simulate natural gut colonization and to test against organisms of unknown acid sensitivity. Normal acid animals received pH 7 formula, no ranitidine. On day 3, the mesenteric lymph nodes (MLN), spleen, liver, and cecum were harvested and cultured. Results: Bacterial growth was inhibited at pH 2 and 3, growth was logarithmic above pH 4 (P <.001). Total and organ-specific translocation was reduced at pH 3 and below in both models (P <.05). Translocation with formula pH 3 equaled normal acid animals. Quantitative cecal colonization was reduced in pups receiving pH 3 and below in both models (:P <.05). Conclusion: Acidification of formula below pH 4 is bactericidal to enteric organisms. Acidified formula decreases bacterial translocation and gut colonization. J Pediatr Surg 36:56-62. Copyright (C) 2001 by W.B. Saunders Company.