Rabbits fed a diet rich in oleic acid develop gallstones consisting of calcium salts of (5α)-glyco-allodeoxycholic acid. To study the metabolic pathway of oleic acid, we followed the changes in plasma, hepatic and biliary lip-ids 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 hypercholes-terolemia. 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 5α-dihydroxy bile acids, increased the proportion of 5β-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 (5α)-glyco-allocholic acid which is secreted into bile and transformed by gut bacteria to form (5α)-allodeoxycholic acid. Kanamycin abolished gallstone formation by inhibiting intestinal bacterial dehydroxylation.