Although beta-oxidation of fatty acids occurs in both peroxisomes and mitochondria, beta-oxidizing enzymes in these organelles have distinct differences in their specifity and sensitivity to inhibitors. In this study, the effects of the phosphodiesterase inhibitor enoximone on hepatic peroxisomal and mitochondrial beta-oxidation were investigated. In liver homogenates from control rats, cyanide-insensitive peroxisomal beta-oxidation of palmitoyl-CoA was inhibited progressively by increasing concentrations of enoximone. Similar results were obtained in liver homogenates from rats pretreated with the known peroxisomal proliferator diethylhexylphthalate. In contrast, mitochondrial beta-oxidation of palmitoyl-CoA was not inhibited by enoximone. These data show that enoximone selectively inhibits basal as well as induced peroxisomal, but not mitochondrial, beta-oxidation of the CoA thioester of long-chain fatty acids. The availability of specific inhibitors of peroxisomal beta-oxidation should prove useful in elucidating regulatory mechanisms operative in this pathway in normal as well as in proliferated peroxisomes.