Abstract The acetyl-CoA-dependent elongation of medium-chain acyl-CoA in the presence of pyridine nucleotide was studied in rat liver. The activity was increased by the administration of peroxisome proliferators, clofibrate and di-(2-ethylhexyl)phthalate, and the change was more remarkable in peroxisomes than in mitochondria. Addition of 0.01% Triton X-100 to the incubation mixture caused an increase in the mitochondrial activity, whereas the peroxisomal activity did not increase significantly. The pH optimum for the peroxisomal activity was in the range of pH 6.5–7.0 and that for the mitochondrial activity was pH 7.5–8.0. The specificities of primer chain length in both organelles were almost the same, and octanoyl-CoA was the preferred substrate. Peroxisomal activity was completely inhibited by the addition of 1 m m N-ethylmaleimide or 1 m m p-hydroxymercuribenzoic acid, while the activity did not change on the addition of 1 m m KCN or an antibody to acyl-CoA oxidase, the first enzyme of the peroxisomal β-oxidation system. The activity of enoyl-CoA reductase, which catalyzes the last step of the elongation system, was also detected in peroxisomes, although the main activity was localized in microsomes. When the liver peroxisomal fraction of clofibrate-treated rats was incubated with a mixture of octanoyl-CoA, acetyl-CoA, NADH, NADPH, and Triton X-100 in a buffer system, dodecanoyl-CoA was detected as the main product by radio-gas chromatography. On the other hand, the elongation activity was decreased greatly by the addition of NAD + into the mixture. These results indicate that (i) peroxisomes have activity to elongate medium chain acyl-CoA; (ii) the peroxisomal elongation system may consist of the reverse reaction of the β-oxidation system except for the last step, which is catalyzed by enoyl-CoA reductase; and (iii) the peroxisomal elongation system is less active than the β-oxidation system under physiological conditions.