Two major glucoamylase isoenzymes (GAI and GAII) have been identified in culture supernatants of Aspergillus awamori. It has been suggested that a stepwise degradation of a native enzyme during the fermentation by proteases and/or glucosidases results in the formation of isoenzymes that have different characteristics concerning substrate specificity and stability to pH and temperature. In this study, the glucoamylase isoenzymes produced by Aspergillus awamori using liquid media with C/N 10 (2.0% starch, 0.45% (NH4)(2) SO4) and C/N 26 (5.2% starch, 0.45% (NH4)(2) SO4) were analyzed. In both cases, GAI and GAII were characterized concerning its hydrolitic activities, mol wt, and isoeletric point. Using HPLC gel filtration and FPLC chromatofocusing, it was obtained for GAI a mol wt of 110,000 Da, pi 3.45 and for GAII a mol wt of 86,000 Da, pi 3.65. A different isoenzymes proportion was observed by the use of the two C/N ratios. In the lower carbohydrate content, fermentation of the GAI form predominated, whereas in the C/N 26 medium, GAII was prevalent. Gel eletrophoresis, amino acid analysis, and structural data confirmed that both preparations were glucoamylases with a high degree of homogeneity.