A common function of peroxisomes in eukaryotic cells is beta-oxidation of fatty acids. In animal cells, beta-oxidation is compartmentalized to peroxisomes and mitochondria. Although regulation of beta-oxidation in mitochondria has been extensively studied, knowledge on its regulation in peroxisomes is still limited. We have considered the possibility that peroxisomes may contain acyl-CoA thioesterases with different substrate specificities that possibly regulate metabolism of different lipids by regulation of substrate availability. In the present study, we have investigated the presence of short-chain and long-chain acyl-CoA thioesterase activities in rat liver peroxisomes. Light-mitochondrial fractions, enriched in peroxisomes, were fractionated by Nycodenz density gradient centrifugation and gradient fractions were analyzed for acyl-CoA thioesterase and marker enzyme distributions. Fractionation of livers from normal rats showed that most of the long-chain acyl-CoA thioesterase activity was localized in microsomes and mitochondria, and only low activity was found in fractions containing peroxisomes. The gradient distribution of propionyl-CoA thioesterase activity showed this activity to be localized mainly in mitochondria and in fractions possibly representing lysosomes, with a small peak of activity in peroxisomal fractions. Di(2-ethylhexyl)phthalate treatment induced the specific propionyl-CoA thioesterase activity approximately threefold in the peak mitochondrial fractions and about onefold in peroxisomal fractions; the activity appeared to be almost exclusively localized to these organelles. The specific activity of myristoyl-CoA thioesterase was induced 1-2-fold in peroxisomal peak fractions and more than 10-fold in the mitochondrial peak fraction, whereas it was unchanged in microsomes. The chain-length specificity of acyl-CoA thioesterase activity in isolated peroxisomes suggests that peroxisomes contain an inducible short-chain thioesterase active on C2-C4 acyl-CoA species (possibly a 'propionyl-CoA' thioesterase). In addition, peroxisomes contain medium-chain to long-chain thioesterase activity, probably due to separate enzymes based on the different chain-length specificities observed in peroxisomes from normal and di(2-ethylhexyl)phthalate-treated rats. A long-chain acyl-CoA thioesterase was partially purified from isolated peroxisomes and found to be active only on fatty-acyl-CoA species longer than octanoyl-CoA. The protein is apparently a monomer of about 40 kDa and clearly different from microsomal long-chain acyl-CoA thioesterase. An induction of this long-chain thioesterase may explain the observed change in chain-length specificity in peroxisomes isolated from normal and di(2-ethylhexyl)phthalate-treated rats. Possible physiological functions of these thioesterases are discussed.