Aspergillus niger glucoamylases GI and GII (E.C. 3.2.1.3) were isolated from a commercial enzyme preparation by ammonium sulfate precipitation followed by DEAE-cellulose ion exchange chromatography. Both enzymes consist of a single glycosylated polypeptide chain. The molecular weights of GI and GII were determined by sedimentation equilibrium ultracentrifugation to 52,000 and 46,000, respectively, and by molecular sieving to 65,000 and 55,000. The amino acid compositions of GI and GII were very similar. Furthermore, the N-terminal amino acid sequence of the intact GI and GII as well as of their cyanogen fragments were identical, suggesting great homology in the primary structure of the two forms. In addition the digests of GI and GII produced respectively by Armillaria mellea protease, Staphylococcus aureus V8 protease, and submaxilla~ protease were analyzed by high pressure gel permeation chromatography. The elution profiles were also consistent with GI and GII having similar polypeptide chains. However, digestion with carboxypeptidase Y showed different C-terminal residues of the two forms. 1. INTRODUCTION Glucoamylase (l,4-aD -glucan glucohydrolase, E.C. 3.2.1.3) catalyzes the release of Dglucose from the non-reducing end of starch, glycogen and gluco-oligosaccharides. Although the ct-i,6-glucosidic linkages are cleaved less readily than the a-l,4-glucosidic linkages (17, 28, 38), the debranching capacity of glucoamylases is sufficient to make them important in the industrial production of glucose from starch, a Abbreviations: GI and GII denote two forms of glucoamylase.