Neopullulanase was the key enzyme to open the door for the formulation of the concept of the aamylasefamily. The enzyme catalyzes both hydrolysis and transglycosylation at α-1, 4- and α-1, 6-glucosidic linkages through one active center. We found a unique macromolecule recognition by the enzyme. A mixture of amylose and amylopectin from various sources was used as the substrate. Neopullulanase completely hydrolyzedamylose to maltose and a small amount of glucose, but scarcely hydrolyzed amylopectin. Although the molecular mass of potato amylopectin (approximately 10⁸ Da) decreased slightly, the degradation of amylopectincompletely halted at the molecular mass of approximately 10⁷Da. This difference in action on two macromolecules was also found in cyclomaltodextrinase and maltogenic amylase from Bacillus licheniformis.