Measurements made by chemiluminescence (CL) were used to estimate effective D- and L-thyroxine concentrations and to study their effects on free-radical oxidation processes in the mitochondrial and synaptosomal fractions of rat cerebral cortex in vitro. These experiments showed that in a model system containing riboflavin, the antioxidant activity of D-T4 was 2.2 times greater than that of L-T4. The effective concentrations for the two forms of thyroxine were I50 = 74.3 +/- 7.1 microM for D-T4 and I50 = 154.7 +/- 12.3 microM for L-T4. Studies of the in vitro effects of thyroxine on the membrane fraction of the cerebral cortex were based on luminol-dependent peroxide CL. At physiological concentrations (10 nM), both isomers of the hormone had identical antioxidant activities, which were stronger in the mitochondrial traction, where the intensity of CL decreased by 69% and 66%, compared with 45% and 46% decreases in the synaptosomal fraction. Since the D-form has no hormonal activity, it is suggested that this effect is associated with the phenolic nature of thyroxine.