The antioxidant effect of melatonin was evaluated by using a flow injection analysis chemiluminescence (FIA-CL) method to measure alone its direct antioxidant behaviour in aqueous solutions. Inhibition by melatonin, DL-tryptophan or serotonin (5-hydroxytryptamine, 5-HT) of the steady CL emissions of various oxidants with luminol was measured. Log IC50 values of melatonin were calculated: -2.6 +/- 0.1 M for superoxide (O-2(.-)), -3.0 +/- 0.02 M, for hydrogen peroxide (H2O2), -4.7 +/- 0.04 M, forhypochlorous anion (-OCL), -2.8 +/- 0.1 M, for hydoxyl ((OH)-O-.) and -2.0 +/- 0.03 M, for peroxyrntrite (ONOO-). Thus melatonin exerted a limited direct antioxidant activity towards reactive oxygen species (ROS) in aqueous solutions, broadly comparable to that of tryptophan, but less than that of serotonin and of major antioxidants such as ascorbic acid. This suggests that where the benefits of melatonin are correctly attributed to antioxidant behaviour, this is mediated largely through antioxidant enzymes. The pharmacological implications are discussed. (C) 2003 Elsevier Ltd. All rights reserved.