Dicarboxylic acids are formed by omega-oxidation of fatty acids in the endoplasmic reticulum and degraded as the CoA ester via beta-oxidation in peroxisomes. Both synthesis and degradation of dicarboxylic acids occur mainly in kidney and liver, and the chain-shortened dicarboxylic acids are excreted in the urine as the free acids, implying that acyl-CoA thioesterases (ACOTs), which hydrolyze CoA esters to the free acid and CoASH, are needed for the release of the free acids. Recent studies show that peroxisomes contain several acyl-CoA thioesterases with different functions. We have now expressed a peroxisomal acyl-CoA thioesterase with a previously unknown function, ACOT4, which we show is active on dicarboxylyl-CoA esters. We also expressed ACOT8, another peroxisomal acyl-CoA thioesterase that was previously shown to hydrolyze a large variety of CoA esters. Acot4 and Acot8 are both strongly expressed in kidney and liver and are also target genes for the peroxisome proliferator-activated receptor alpha. Enzyme activity measurements with expressed ACOT4 and ACOT8 show that both enzymes hydrolyze CoA esters of dicarboxylic acids with high activity but with strikingly different specificities. Whereas ACOT4 mainly hydrolyzes succinyl-CoA, ACOT8 preferentially hydrolyzes longer dicarboxylyl-CoA esters (glutaryl-CoA, adipyl-CoA, suberyl-CoA, sebacyl-CoA, and dodecanedioyl-CoA). The identification of a highly specific succinyl-CoA thioesterase in peroxisomes strongly suggests that peroxisomal beta-oxidation of dicarboxylic acids leads to formation of succinate, at least under certain conditions, and that ACOT4 and ACOT8 are responsible for the termination of beta-oxidation of dicarboxylic acids of medium-chain length with the concomitant release of the corresponding free acids.