The long-chain fatty acid composition in rat liver, by which peroxisomal beta-oxidation system was induced, was analyzed in vitro and in vivo. In primary culture of hepatocytes, treatment with 0.4 mM clofibrate or long-chain fatty acids caused an increase in the peroxisomal beta-oxidation activity, in addition the oleic acid [18:1(n-9)] content in the cells was increased two-fold by addition of very long-chain fatty acids to the culture medium, but clofibrate had no effect. A two-fold of increase in 18:1(n-9) content was observed in the hepatic subcellular fractions from rats fed a diet containing 0.25% clofibric acid. The ratio of cis-vaccenic acid [18:1(n-7)] to total 18:1 content decreased by more than half compared with control in clofibric acid treated liver. The level of 18:1(n-7) was not changed in all experiments. It is suggested that perturbation of C-18 chain length fatty acids occurs in peroxisomal beta-oxidation induced liver.