Changes in membrane surface properties of hepatic peroxisomes of rats under several conditions were observed by aqueous polymer two-phase systems, which contained 6% (w/w) dextran T 500, 6% (w/w) polyethyleneglycol 4000, 250 mmol sucrose/kg and various concentrations of sodium phosphate buffer. The partition of peroxisomes into the upper phase depended to a large extent on their membrane surface charge. The cross-points of peroxisomes shifted from 5.55 to 5.25 and 5.2 after the administration of clofibrate and aspirin for 2 weeks, respectively, although that of alloxan-diabetic rat peroxisomes was not altered. The hydrophobic properties of peroxisomes, examined by means of a partition containing polyethyleneglycol monostearate, were altered by diabetes and starvation, but no change occurred in rats treated with clofibrate or aspirin. In the liver of rats fed a high-fat diet, the partition of peroxisomes was the same as that of the control. These findings indicate that hypolipidemic drugs such as clofibrate and aspirin induce the proliferation of peroxisomes and lead to the alteration of the surface charge of peroxisomal membranes. Diabetes or fasting lead to an alteration mainly of the hydrophobic properties. Both changes are probably due to alteration of content and/or composition of the proteins and the phospholipids in peroxisomal membrane under the conditions used.