Three coenzyme A (CoA) molecular species, i.e., acetyl-CoA, malonyl-CoA, and nonesterified CoA (CoASH), in 13 types of fasted rat tissue were analyzed. A relatively larger pool size of total CoA, consisting of acetyl-CoA, malonyl-CoA, and CoASH, was observed in the medulla oblongata, liver, heart, and brown adipose tissue. Focusing on changes in the CoA pool size in response to the nutrient composition of the diet given, total CoA pools in rats continuously fed a high-fat diet for 4weeks were significantly higher in the hypothalamus, cerebellum, and kidney, and significantly lower in the liver and skeletal muscle than those of rats fed a high-carbohydrate or high-protein diet. In particular, reductions in the liver were remarkable and were caused by decreased CoASH levels. Consequently, the total CoA pool size was reduced by approximately one-fifth of the hepatic contents of rats fed the other diets. In the hypothalamus, which monitors energy balance, all three CoA molecular species measured were at higher levels when rats were fed the high-fat diet. Thus, it was of interest that feeding rats a high-fat diet affected the behaviors of CoA pools in the hypothalamus, liver, and skeletal muscle, suggesting a significant relationship between CoA pools, especially malonyl-CoA and/or CoASH pools, and lipid metabolism in vivo.