Glucoamylase produced by Aspergillus niger was observed to increase in culture media at two specific intervals, namely, after three days and after six days of incubation. The lowest levels of enzyme production occurred when the nitrogen source was asparagine, trypticase or urea. Intermediate quantities of enzyme were formed when the fungi were grown on a nitrogen source of yeast extract. Corn steeping liquor and nutrient broth (2.5%) gave highest enzyme activity. The inorganic nitrogen source providing the best glucoamylase yield was ammonium-N. Nitrate-N or a mixture of ammonium-N and nitrate-N was less effective. When inorganic nitrogen was employed, the two periods of glucoamylase production were not as readily demonstrated. With ammonium-N, all the glucoamylase is produced during the fourth or fifth day of incubation, thus showing only one period of enzyme synthesis. The quantity of glucoamylase produced depended upon the monosaccharide added to the growth medium. Glucose yielded the highest level of enzyme regardless of the nitrogen sources. Mannose, usually, was slightly more effective than sorbitol and produced about half the quantity of glucoamylase obtained from glucose. Lowest levels of enzyme occurred when fructose or xylose served as carbon sources. Growth on 1.0 or 2.0% carbohydrate, rather than 5.0%, was found to result in higher levels of glucoamylase. Although A. niger NRRL 330 was the principle organism tested, the characteristics of glucoamylase synthesis were not specific for this strain since similar results were obtained with A. niger NRRL 3122 and A. awamori NRRL 3112. © 1969, Applied Microbiology, Molecular and Cellular Biosciences Research Foundation. All rights reserved.