Inhibition of the overt mitochondrial carnitine palmitoyltransferase by malonyl-CoA is important in the regulation of fatty acid oxidation. In the past, the contribution of peroxisomal carnitine acyltransferase activity to the generation of medium- and long-chain acylcarnitines in the cytoplasm has been ignored. On the basis of marker enzyme levels, we now estimate that peroxisomal palmitoyltransferase activity constitutes about 20% of the peroxisomal plus overt-mitochondrial pool in fed rat liver. When assayed in situ, both the palmitoyltransferase and decanoyltransferase activities of gradient-purified peroxisomes are sensitive to malonyl-CoA, with up to 90% inhibition reached at less than 10 microM-malonyl-CoA. Very similar results were obtained with intact gradient-purified mitochondria from the same livers. In addition, the acyl-CoA substrate chain-length specificity was identical in both the peroxisomes and the mitochondria, with a decanoyltransferase/palmitoyltransferase ratio of 2. Thus the overt carnitine acyltransferase activities in peroxisomes and mitochondria have the same properties. Further, the malonyl-CoA sensitivity of the peroxisomal activity is lost on solubilization, as has been observed for the overt mitochondrial enzyme. It is suggested that malonyl-CoA inhibition of the peroxisomal enzyme as well as of the mitochondrial enzyme is important for the regulation of mitochondrial fatty acid oxidation.