This paper describes the fatty acid metabolism of liver mitochondria as determined by I-(14)C acetate incorporation in rats which were fed a diet supplemented with cholesterol, cholic acid and stearic acid (CCS). In liver mitochondria, fatty acid biosynthesis was considered to be the main part of lipid biosynthesis and cholesterol biosynthesis to be negligible. Fatty acid biosynthesis of the liver mitochondria did not change by the addition of ATP and NADH, but decreased by the addition of palmitoyl carnitine. The recovery of (14)CO(2) from I-(14)C acetate in the mitochondria was significantly increased by the addition of ATP. The CCS feeding markedly elevated cholesterol and phospholipid levels. As for the serum fatty acid composition, stearic acid and oleic acid increased, and linoleic acid and arachidonic acid decreased. On the other hand, in liver mitochondria, oleic acid increased and arachidonic acid decreased. The CCS feeding had a tendency to increase fatty acid biosynthesis of liver mitochondria although there was no significant difference in the pattern of each fatty acid biosynthesis between the CCS and control groups.