Nystose represents a fructooligosaccharide with two fructose molecules linked via beta(1----2) bonds to the fructosyl moiety of sucrose. This tetrasaccharide was subjected to an array of in vitro tests designed for the assessment of potential sugar substitutes before animal or human studies. beta-Fructosidase from yeast cleaved nystose at about 5% of the initial rate observed with sucrose. The terminal fructose was released first. Glycosyltransferase from Streptococcus mutans #620 did not utilize nystose for the formation of a glucan-type polysaccharide. Anaerobic fermentation of nystose by a suspension of mixed dental plaque microorganisms and by S. mutans NCTC 10449 was about half as fast as with sucrose. Thin-layer chromatography at various reaction times with S. mutans NCTC 10449 indicated the terminal fructose as the site of first attack. Analyses for free monosaccharides confirmed these data because free fructose exceeded free glucose at early reaction times far more than would follow from the 3:1 ratio of fructose to glucose in the nystose molecule. High pressure liquid chromatography assays demonstrated lactic and acetic acids as the main fermentation products. Carbohydrases from human jejunal mucosa did not attack nystose. However, cecal anaerobic microorganisms of the rat fermented nystose rapidly into acids.