To understand the possible role of salicylic acid in the pathogenesis of Reye's syndrome, we examined its effect on the oxidative metabolism of fatty acids in the rat liver mitochondrial-peroxisomal fraction. Fatty acids of different chain lengths are oxidized in different organelles. Octanoic acid is oxidized in mitochondria, lignoceric acid in peroxisomes, and palmitic acid in both mitochondria and peroxisomes. Salicylic acid (up to 1 mM concentration) had no effect on the oxidation of [1-14C]lignoceric acid. However, at the same concentration it inhibited the oxidation of [1-14C]palmitic acid by 26% and [1-14C] octanoic acid by 42%. The apparent Ki for the oxidation of [1-14C] octanoic acid, [1-14C]palmitic acid and [1-14C]lignoceric acid were 0.27, 6.0, and 14.8 mM, respectively. This selective inhibition of mitochondrial oxidation of medium-chain (octanoic acid) and long-chain (palmitic acid) fatty acids by salicylic acid may potentiate the accumulation of fatty acids in plasma in Reye's syndrome patients.