The contribution of peroxisomes to palmitate beta-oxidation in rat heart was estimated by either inhibiting mitochondrial beta-oxidation or measuring the activity of acyl-CoA oxidase. When respiratory inhibitors such as KCN or antimycin plus rotenone, or inhibitors of mitochondrial fatty acid uptake such as 2-tetradecylglycidic acid or 2-bromopalmitate, were used, degrees of inhibitions ranging from 24% to 87% were observed for palmitate beta-oxidation by a rat heart homogenate. Although the oxidation of palmitoyl-L-carnitine by coupled rat heart mitochondria was almost completely (94%) inhibited by KCN, the inhibition by antimycin plus rotenone was incomplete (77%) and was stimulated by L-carnitine. A direct assay of acyl-CoA oxidase, based on the spectrophotometric measurement at 300 nm of 2,4-decadienoyl-CoA formation from 4-trans-decenoyl-CoA, was evaluated with the aim of obtaining reliable values for the activity of this enzyme, which is presumed to catalyse the rate-limiting step of peroxisomal beta-oxidation. Activities determined by use of this assay were much higher than activities obtained by a coupled assay [Small, Burdett and Connock (1985) Biochem. J. 227, 205-210] commonly used to measure the activity of acyl-CoA oxidase. However, both methods yielded the same relative activities with different tissue homogenates. Based on an estimated palmitoyl-CoA oxidase activity of 0.3 nmol/min per mg of protein, the contribution of peroxisomes to palmitate beta-oxidation in a rat heart homogenate would optimally be 4%, and most likely is several-fold lower.