Rats were fed a 70% carbohydrate, 70% protein, 70% fat, or a standard purified diet for 7 d to determine the effect of the diet on heart glycogen synthase response to an acute insulin challenge. Rats fed the high protein or the high fat diets, i.e., the carbohydrate-free diets, exhibited insulin resistance as evidence by higher plasma glucose levels following insulin administration when compared to rats fed high carbohydrate or standard diet. The diets had no effect on the initial proportion of synthase in the active or I form. Insulin injection resulted in an increase in the proportion of synthase in the active form in rats fed the standard, high carbohydrate or high protein diets, but not in rats fed the high fat diet. Synthase phosphatase activity was similar in rats fed one of the four diets compared to rats fed a nonpurified diet. Thus the lack of synthase response to insulin in fat-fed rats was not due to diminished synthase phosphatase activity. Neither the diets nor insulin administration had any effect on the proportion of phosphorylase in the active or a form. Cardiac glycogen was significantly lower in rats fed the high fat diet than in those fed the standard diet. The latter was a surprising observation since the high fat diet was used to simulate a starved state and cardiac glycogen concentrations increase with starvation.