Membranes were prepared from peroxisomes which had been isolated from the livers of normal mice and from mice treated with clofibrate (a hypolipidemic drug and peroxisome proliferator). Phospholipid analysis of these membranes revealed that clofibrate treatment resulted in a decrease in the membrane content of phosphatidylcholine, the most abundant phospholipid, and a concomitant increase in the amount of lysophosphatidylcholine, this latter component reaching a level of almost 6% of the total membrane phospholipid. The concentrations of other phospholipids in these membranes were not significantly altered. The parallel analysis of microsomal membranes demonstrated an analogous increase in the level of lysophosphatidylcholine following clofibrate treatment. In control experiments with microsomal membranes employing quinacrine, an inhibitor of phospholipase A2, the increased lysophosphatidylcholine concentration was still observed in clofibrate-treated animals. As well, a decrease in the proportion of microsomal phosphatidylcholine with clofibrate treatment was seen when quinacrine was used. Fatty acid analysis of the phosphatidylcholines from peroxisomal membranes showed some minor changes, including an increase in one component tentatively identified as docosahexaenoic acid, in clofibrate-treated animals. Overall, these data demonstrate that clofibrate causes a marked perturbation of the phospholipid composition of peroxisomal membranes, and are interpreted as indicating that the main site of action of the drug is the deacylation-reacylation cycle between phosphatidylcholine and lysophosphatidylcholine.