Abstract Acyl-coenzyme A (CoA) hydrolases/thioesterases are a group of enzymes that catalyze the hydrolysis of acyl-CoA thioesters to free fatty acids and CoA-SH. The potency of these enzymes may serve to modulate intracellular concentrations of acyl-CoAs, free fatty acids, and CoA to affect various cellular functions, including lipid metabolism. In this study, we investigated the effect of diabetes and fasting on the protein levels of mitochondrial (MTE-I) and cytosolic acyl-CoA thioesterases (CTE-I), multigene family members of this class of enzymes, in adult rat liver. Rats were treated with alloxan to induce diabetes or fasted for 72 hours. Western blot analysis with the liver homogenates revealed 2.8-fold and 3.8-fold increases in MTE-I and 8.5-fold and 9.2-fold increases in CTE-I under the diabetic and fasting conditions, respectively, compared with the control in which the level of MTE-I was 4.3-fold higher than CTE-I. Serum level of free fatty acids was elevated 5-fold and 2.5-fold in diabetic and fasted rats, respectively. These results confirm the adaptive induction of MTE-I and CTE-I in response to fatty acid overload in the liver, being consistent with their auxiliary role in fatty acid degradation.