The effect of gemfibrozil, a peroxisome proliferator, on lipid biosynthesis from acetyl-CoA derived from peroxisomal beta-oxidation was studied. The specific activity of the peroxisomal fatty acyl-CoA beta-oxidation system of rats fed a chow containing 0.2% gemfibrozil for 2 weeks was approximately five times higher than that of control rats. When [1-14C]lignoceric acid, a very-long-chain fatty acid which is degraded exclusively by the peroxisomal beta-oxidation system at first, was injected into rats treated with gemfibrozil, radioactivity and content of bile acid in the bile were enhanced to approximately 2.2 and 3.5 times the control, respectively. Gemfibrozil increased the radioactivity and content of chenodeoxycholic acid more than that of cholic acid. The incorporation of radioactivity into cholesterol in the bile was as much as 4.5 times greater than the control, and content was 2.6 times greater. In the liver, incorporation of [14C]lignoceric acid into the simple lipids phosphatidylethanolamine and phosphatidylcholine was unaffected by gemfibrozil. The radioactivity and content of cholesterol separated from the simple lipids were also virtually unaffected. However, the specific activities of 3-hydroxy-3-methylglutararyl-CoA reductase (rate-limiting enzyme of cholesterol synthesis) of peroxisomes and microsomes were remarkably stimulated by gemfibrozil treatment. These results suggest that biosyntheses of cholesterol and bile acid from acetyl-CoA derived from peroxisomal beta-oxidation are stimulated by gemfibrozil, due at least in part to activation of the peroxisomal beta-oxidation system and 3-hydroxy-3-methylglutaryl-CoA reductase of peroxisomes and/or microsomes. Most peroxisomal proliferators (e.g. clofibrate) have been known to inhibit 3-hydroxy-3-methylglutaryl-CoA reductase activity. Therefore, gemfibrozil is expected to be a very useful tool for elucidating the relationship between peroxisomes and the biosyntheses of cholesterol and bile acid.