Acyl-CoA oxidase, the first enzyme of the peroxisomal beta-oxidation, was proved to be rate-limiting for this process in homogenates of rat liver, kidney, adrenal gland, heart and skeletal muscle. Acyl-CoA oxidase activity, based on H2O2-dependent leuko-dichlorofluorescein oxidation in tissue extract, was compared with radiochemically assayed peroxisomal beta-oxidation rates. Dichlorofluorescein production was a valid measure of peroxisomal fatty acid oxidation only in liver and kidney, but not in adrenal gland, heart or skeletal muscle. Production of 14C-labeled acid-soluble products from 1-14C-labeled fatty acids in the presence of antimycin-rotenone appears to be a more accurate and sensitive estimate of peroxisomal beta-oxidation than the acyl-CoA oxidase activity on base of H2O2 production. Chain-length specificity of acyl-CoA oxidase changed with the acyl-CoA concentrations used. Below 80 microM, palmitoyl-CoA showed the highest activity of the measured substrates in rat liver extract. No indications were obtained for the presence in rat liver of more forms of acyl-CoA oxidase with different chain-length specificity.