Bifidobacterium adolescentis ATCC 15703, Bifidobacterium breve ATCC 15700, Bifidobacterium longum ATCC 15707, and human fecal microbiota were cultivated in vitro with d-xylose, l-arabinose, xylo-oligosaccharides (XOS), and arabinoxylo-oligosaccharides (AXOS) as carbon sources. The pH, formation of volatile fatty acids, and carbohydrate utilization profiles were followed. In the pure bifidobacteria cultures optical density and in the fecal slurries pressure and H(2) were also detected. A differing substrate preference was observed among the various bifidobacteria strains. B. adolescentis grew on XOS, slowly on d-xylose, but not on l-arabinose. In contrast, B. longum preferred l-arabinose and did not grow on pure d-xylose or XOS. Both strains were able to utilize AXOS but with differing strategies, since after the cleavage of l-arabinose B. adolescentis consumed the XOS formed, whereas B. longum fermented the l-arabinose released. B. breve grew poorly on all of the substrates provided. A bifidobacterial mixture and the fecal microbiota were able to utilize pure singly substituted AXOS almost completely, but pure AXOS with a doubly substituted xylose residue was fermented only by the fecal microbiota. Thus, AXOS appear to be potential candidates for slowly fermenting prebiotics, but their prebiotic effects may be dependent on the type of arabinose substitution and the presence of other carbohydrates.