The pathogenesis of neonatal necrotising enterocolitis (NEC) remains unclear. Gnotobiotic quails fed a lactose diet have been used to investigate the role of clostridial strains originating from faecal specimens of neonates through the intestinal lesions, the changes in microflora balance and the production of bacterial metabolites, i.e., short-chain fatty acids and hydrogen. Bifidobacteria are thought to exert various beneficial effects on host health, including interaction with the colonic microflora. Therefore, it was hypothesised that a protective role could be exercised through bifidobacterial colonisation. A Clostridium butyricum strain (CB 155-3) and a whole faecal flora including three clostridial species (C. butyricum, C. perfringens, C. difficile), each from premature infants suffering from NEC, caused caecal lesions in quails similar to those observed in man, i.e., thickening of the caecal wall with gas cysts, haemorrhagic ulceration and necrotic areas. Conversely, a whole faecal flora including bifidobacteria (identified as Bifidobacterium pseudo-catenulatum) and no clostridia, isolated from a healthy premature infant, was unable to produce NEC-like lesions. When the two clostridial groups were associated with a Bifidobacterium strain (B. infantis-longum, CUETM 89-215, isolated from a healthy infant), bifidobacterial colonisation suppressed all pathological lesions. This study is the first demonstration of a protective role for bifidobacteria against NEC via the inhibition of growth of C. butyricum or the disappearance of C. perfringens. C. difficile was not found to be responsible for the aetiology of the caecal lesions in quails. The main effect of bifidobacteria on lactose fermentation was either a dramatic decrease or a disappearance of butyric acid. The protective role was not associated with changes in H2 production. Therefore, a new step between colonic colonisation and its relevance to NEC is thought to involve the fermentation of unabsorbed lactose into butyric acid at the onset of the disease.