Low-molecular-mass beta-(2,6)-linked fructose-oligosaccharides (beta-(2,6)-FOS) were examined as a new carbohydrate source for growth of bifidobacteria. beta-(2,6)-FOS were prepared from microbial high-molecular-mass levan by acid hydrolysis and refined by cation-exchange chromatography. (13)C-NMR spectroscopy confirmed the presence of predominantly beta-(2,6)-fructosyl linkages in the oligosaccharides. More than 80% beta-(2,6)-FOS was recovered after in vitro incubation with amylolytic and proteolytic enzymes, implying resistance to degradation in the upper intestinal tract. Bifidobacterium adolescentis, B. longum, B. breve, and B. pseudocatenulatum were studied in vitro for their ability to metabolize beta-(2,6)-FOS. Growth, decrease in pH, formation of short- chain fatty acids (lactate, acetate, formate) and degradation of beta-(2,6)-FOS were markedly different among species. B. adolescentis showed the best growth, produced the highest amounts of organic acids and metabolized both short- and long-chain beta-(2, 6)-FOS.