Alternanase, an endoglucanase that hydrolyzes the bacterial exopolysaccharide alternan, will also hydrolyze the trisaccharide, panose, to produce glucose and a disaccharide that can be formed into a novel, cyclic tetrasaccharide. The glucose can then be selectively and quantitatively measured by enzyme-based reaction which forms the basis of a coupled enzyme assay to quantitate alternanase activity. By this method a preparation of alternanase purified by affinity chromatography on immobilized isomaltose had a maximum reaction rate (V-max of 0.75 mumol glucose min(-1) and a K-m of 34 mM for panose. Two competitive inhibitors of alternanase activity were also evaluated using this coupled enzyme assay: isomaltose had a K-i of 94 mM while the cyclic tetrasaccharide had a K-i of 66 mM.