Acyl-CoA thioesterases are a group of enzymes that catalyze the hydrolysis of acyl-CoAs to the free fatty acid and coenzyme A (CoASH), providing the potential to regulate intracellular levels of acyl-CoAs, free fatty acids and CoASH. These enzymes are localized in almost all cellular compartments such as endoplasmic reticulum, cytosol, mitochondria and peroxisomes. Acyl-CoA thioesterases are highly regulated by peroxisome proliferator-activated receptors (PPARs), and other nutritional factors, which has led to the conclusion that they are involved in lipid metabolism. Although the physiological functions for these enzymes are not yet fully understood, recent cloning and more in-depth characterization of acyl-CoA thioesterases has assisted in discussion of putative functions for specific enzymes. Here we review the acyl-CoA thioesterases characterized to date and also address the diverse putative functions for these enzymes, such as in ligand supply for nuclear receptors, and regulation and termination of fatty acid oxidation in mitochondria and peroxisomes.