We have investigated the effects of peroxisome proliferators on rat liver long-chain acyl-CoA thioesterase activities. Subcellular fractionations of liver homogenates from control, clofibrate- and di(2-ethylhexyl)phthalate-treated rats confirmed earlier studies which demonstrated that peroxisome-proliferating drugs induce long-chain acyl-CoA thioesterase activity mainly in the mitochondrial and cytosolic fractions. The aim of the present study was to investigate whether the induced activities were due to increases in normally expressed enzymes, or due to induction of novel enzymes. To investigate whether structurally different peroxisome proliferators differentially induced thioesterase activities, we tested the effects of di(2-ethylhexyl)phthalate (a plastisizer) and the hypolipidemic drug clofibrate. For this purpose, we established an analytical size exclusion chromatography method. Chromatography of solubilised mitochondrial matrix proteins showed that the activity in control mitochondria was mainly due to enzymes with molecular masses of about 50 kDa and 35 kDa. The activity in samples prepared from clofibrate- and di(2-ethylhexyl)phthalate-treated rats eluted as proteins of about 40 kDa and 110 kDa. Highly purified peroxisomes contained two peaks of activity, which were not induced, that corresponded to molecular masses of 40 kDa and 80 kDa. The 80-kDa peak was shown to be due to dimerization by addition of glycerol. Chromatography of cytosolic fractions from control rat livers indicated the presence of long-chain acyl-CoA thioesterases with molecular masses of approximately 35 kDa and 125 kDa and a broad peak corresponding to a high-molecular-mass protein. The activity in cytosolic fractions from peroxisome-proliferator-treated rats eluted mainly as peaks corresponding to 40, 110 and 150 kDa. In addition, in the 110-kDa peak, a different degree of induction and different chain-length specificities were caused by clofibrate and di(2-ethylhexyl)phthalate, suggesting that these peroxisome proliferators differentially regulate the cytosolic acyl-CoA thioesterase activities. Western blot analysis showed that enzymes in the 40-kDa peak of the peroxisomal and cytosolic fractions were structurally related, but not identical, to a 40-kDa mitochondrial very-long-chain acyl-CoA thioesterase. Our data show that the increased acyl-CoA thioesterase activities in mitochondria and cytosol were mainly due to induction of acyl-CoA thioesterases which are not, or only weakly, expressed under normal conditions.