Rats fed a high-saturated fat diet consumed more energy, gained more weight, and displayed hyperinsulinemia (P < .05) without an elevation in the fasting plasma glucose level, compared with animals on two different high-carbohydrate diets. The total fatty acyl-coenzyme A (CoA) concentration was 18% (P < .0001) and 46% (P < .0001) higher in liver and skeletal muscle, respectively, from rats fed the high-fat diet compared with each of the other diet groups. Major long-chain fatty acyl-CoA molecular species of both tissues in high fat-fed rats reflected the fatty acid profile of the diet. Approximately 29%, 21%, and 16% of total liver and skeletal muscle fatty acyl-CoAs were comprised of oleoyl-CoA, palmitoyl-CoA, and stearoyl-Coa, respectively. The amounts of these three fatty acyl-CoA esters were significantly higher in liver and skeletal muscle after high-fat feeding than with the other diet treatments (P < .0001). In contrast, the concentration of linoleoyl-CoA was lower in both tissues after high-fat feeding (P < .0001). In rats fed the high-fat diet, plasma insulin levels were significantly correlated with gain in body weight or body weight (r = .80, P < .001 for insulin and gain in body weight; r = .73, P < .001 for insulin and body weight). Total fatty acyl-CoA ester content in liver and skeletal muscle was also strongly correlated with the plasma insulin concentration in high fat-fed rats (r = .80, P < .001 for liver; r = .78, P < .001 for skeletal muscle). Regardless of diet treatments, a positive correlation between tissue fatty acyl-CoA level and gain in body weight or body weight was reported from all experimental rats (r = .75, P < .001 for hepatic total acyl-CoA and body weight or weight gain; r = .73, P < .001 for muscle acyl-CoA content and body weight; r = .77, P < .001 for muscle total acyl-CoA and weight gain). These results clearly demonstrate that the dietary obesity induced by altering the macronutrient proportion of the diet from predominantly carbohydrate to predominately fat causes a marked increase in tissue fatty acyl-CoA levels, suggesting the greater utilization of fatty acids associated with high-fat intake. The inhibition of glucose metabolism by substrate competition, termed the glucose-fatty acid cycle, leads to the development of insulin resistance. Tissue fatty acyl-CoA composition could serve as an indicator or could be a causal factor in the development of insulin resistance and hyperinsulinemia.