Degradation of the peroxisomal enzymes fatty acyl-CoA oxidase and catalase was studied in hepatocytes isolated from rats treated with clofibrate and from control rats. Hepatocytes were incubated in the absence of amino acids in order to ensure maximal flux through the autophagic pathway and in the presence of cycloheximide to inhibit protein synthesis. (1) Degradation of the two peroxisomal enzymes in hepatocytes from clofibrate-fed rats, but not in hepatocytes from control rats, was much faster than that of other intracellular enzymes. This increased degradation of the peroxisomal enzymes was almost completely prevented by 3-methyladenine, an inhibitor of macroautophagic sequestration. (2) The increased degradation of the peroxisomal enzymes was also inhibited by a long-chain (C16:0) and a very-long-chain (C26:0) fatty acid, but not by C12:0, a medium-chain fatty acid, or by C8:0, a short-chain fatty acid. These results provide direct evidence for the proposal that autophagic sequestration can be highly selective [(1987) Exp. Mol. Pathol. 46, 114-122]. It is concluded that preferential autophagy of peroxisomes is prevented when these organelles are supplied with their fatty acid substrates.