Incubation of hepatocytes from diabetic rats with glucose results in the translocation of glycogen synthase from soluble fractions to fractions which sediment at 9200 g. The extent of the translocation correlates positively with the intracellular concentration of glucose 6-phosphate. No difference was found between healthy and diabetic rats in the capacity of glycogen synthase to translocate to pellets in response to an increase in glucose 6-phosphate. In diabetic hepatocytes, glycogen synthase in the supernatant fractions was not activated upon incubation of the cells with glucose, whereas this sugar was able to activate the enzyme found in the fractions that could be pelleted. In the 9200-g pellets, the glycogen synthase activity ratio (-glucose 6-phosphate/+glucose 6-phosphate) from both healthy and diabetic animals correlated with the intracellular glucose 6-phosphate levels. In the supernatants, the glycogen synthase activity ratio from healthy cells also correlated with glucose 6-phosphate levels. In contrast, in diabetic cells the activation state of the soluble enzyme remained essentially unchanged despite the accumulation of glucose 6-phosphate.