Rabbits fed a diet rich in oleic acid develop gallstones consisting of calcium salts of (5 alpha)-glyco-allodeoxycholic acid. To study the metabolic pathway of oleic acid, we followed the changes in plasma, hepatic and biliary lipids in this animal model. In addition, to also determine the role played by intestinal microflora on biliary lipid metabolism, we added kanamycin to the oleic acid diet. Oleic acid-fed rabbits rapidly developed hypercholesterolemia. This was associated with an increase in liver 3-hydroxy-3-methylglutaryl coenzyme A reductase activity, accumulation of cholesterol as well as cholestanol in the liver and progressive saturation of cholesterol in bile. [14C]oleic acid fed orally to rabbits was recovered in liver extracts as both cholesterol and cholestanol. With oleic acid feeding, there was a progressive increase in glyco-allodeoxycholic acid culminating in the formation of gallstones. Kanamycin supplement to the oleic acid diet resulted in the same changes in plasma and hepatic sterol metabolism compared with oleic acid-fed rabbits. There was, however, a striking difference in the biliary bile acid profile. Kanamycin supplementation dramatically reduced the proportion of 5 alpha-dihydroxy bile acids, increased the proportion of 5 beta-trihydroxy bile acids and completely abolished gallstone formation. We postulate that, in the rabbit, oleic acid is used as a carbon source for cholesterol synthesis, and a high oleic acid diet increases hepatic cholesterogenesis. Hepatic cholesterol is then metabolized to form cholestanol, followed by (5 alpha)-glyco-allocholic acid which is secreted into bile and transformed by gut bacteria to form (5 alpha)-allodeoxycholic acid. Kanamycin abolished gallstone formation by inhibiting intestinal bacterial dehydroxylation.