Administration of large doses of insulin to intact rats has been shown to stimulate cardiac glycogen synthase phosphatase activity. This results in an activation of glycogen synthase. However, whether a more physiologic stimulus for insulin secretion also resulted in activation of synthase had not been studied. In the present study, rats were fed glucose, casein hydrolysate, or a mixed meal after a 24-hour fast as a means of physiologically stimulating insulin secretion. Lard was fed as a noninsulinotropic nutrient for comparative purposes. Plasma insulin was significantly increased by 15 minutes in rats given a mixed meal or glucose, but surprisingly, no change was observed in rats fed casein hydrolysate. As expected, no change in plasma insulin was observed in rats fed lard. Synthase phosphatase activity was stimulated in rats fed a mixed meal, glucose, or casein hydrolysate, but not in rats fed lard. Likewise, the proportion of synthase in the active (I) form was significantly increased in rats fed a mixed meal, glucose, or casein hydrolysate, but not in rats fed lard. The increase in phosphatase activity and the increased proportion of synthase in the active form following ingestion of casein hydrolysate was unexpected since this occurred in the absence of an increase in insulin. The proportion of phosphorylase in the active (a) form decreased in rats fed glucose, but remained unchanged in rats fed a mixed meal, casein hydrolysate, or lard. The cardiac glycogen concentration decreased dramatically in rats fed casein hydrolysate. Following the other meals, there was either no change or the decrease was modest. Thus, casein hydrolysate ingestion caused a decrease in cardiac glycogen in the absence of a change in the proportion of phosphorylase in the active (a) form, and in the presence of an increase in the proportion of synthase in the active (I) form. The mechanisms involved in these casein hydrolysate stimulated events are unknown.