The glycogen content of yeast rises dramatically just before the onset of the stationary phase of growth. Concomitantly, a rapid increase was found in the glucose 6-phosphate-independent (I) activity of glycogen synthetase, as well as in the total amount of enzyme. A mutant (GS 1-36) was obtained, which did not accumulate glycogen during growth. The synthetase from this strain was in the glucose 6-phosphate-dependent (D) form at all times. The total enzymatic activity of the mutant also increased sharply at the end of the logarithmic phase, although its maximal value was only one third that of the parent strain. Incubation with glucose of wild type resting cells from the logarithmic phase resulted in a slow accumulation of glycogen, which was accelerated after 20 min. At the same time a transformation from the D to I form of the enzyme was detected. The same slow initial rate of glycogen deposition was found with stationary cells of mutant GS 1-36, but the rate gradually declined to zero, rather than accelerating. The interconversion of the I and D forms was obtained with extracts from cells harvested during different phases of growth. Examination of the properties of the I and D forms showed that the latter was much more strongly inhibited by ATP at low glucose 6-phosphate concentration. These findings clearly establish the fundamental role of the I form in glycogen accumulation. When taken together with previous results, they also show that the physiological significance of the I-D interconversion depends on the concentration of glucose 6-phosphate. Under certain conditions glucsoe 6-phosphate appears to regulate directly the activity of the predominant form of the enzyme.