A novel 3-oxacyl-CoA thiolase was found in rat liver. This thiolase, mitochondrial general 3-oxacyl-CoA thiolase and acetoacetyl-CoA thiolase were purified from the rat liver after the induction of these activities by the administration to rats of di(2-ethylhexyl)phthalate, which enhanced the peroxisomal beta-oxidation activity. The new 3-oxoacyl-CoA thiolase was distinguished from mitochondrial and cytoplasmic thiolases by the following: DEAE-cellulose chromatography, phosphocellulose chromatography, immunochemical titration, and substrate specificity. Subcellular fractionation of liver and sucrose-density-gradient centrifugation of the light mitochondrial fraction revealed that the new thiolase was mainly in peroxisomes.