Activity of amylopullulanase from Thermoanaerobacter ethanolicus 39E on alpha-1,6 and alpha-1,4-glucosidic linkages in highly branched mammalian glycogen was analyzed by paper chromatography and C-13 nuclear magnetic resonance (NMR) spectroscopy. Paper chromatography analysis showed that the glycogen hydrolysate consisted of glucose, maltose, maltotriose and maltotetraose, NMR spectroscopy confirmed that no hydrolysate products of alpha-1,6 linkage were present resulting from treatment with the amylopullulanase. Therefore, the amylopullulanase efficiently hydrolyzed glycogen both at alpha-1,6- and at alpha-1,4-glucosidic linkages into oligosaccharides.