Abstract: Antioxidant function of melatonin is well established. However, N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK), a melatonin metabolite is a sparingly investigated biogenic amine, especially in relation to its in vivo antioxidant function. We have evaluated the oxidative damage to biomolecules (DNA, protein and lipid) induced by X-irradiation in C57BL mice and the prophylactic action of AFMK. The extent of DNA damage was analyzed by single-cell gel electrophoresis in cerebral cortex and serum 8-hydroxydeoxyguanosine (8-OHdG) levels by enzyme-linked immunosorbent assay. Oxidative modification of protein and lipid was measured in the terms of carbonyl content and 4-HAE + MDA (4-hydroxyalkenal + malondialdehyde) status of brain cortex. Radiation exposure dramatically augmented the level of 8-OHdG in serum as well as DNA migration in the comet tail. AFMK pretreatment significantly inhibited DNA damage. In addition, radiation-induced augmentation of protein carbonyl content and HAE + MDA was ameliorated by AFMK pretreatment. Whole-body exposure of mice to X-irradiation also reduced the level of brain sulfhydryl contents (protein-bound sulfhydryl, total sulfhydryl, and nonprotein sulfhydryl) which were significantly protected by AFMK. Radiation-induced decline in the total antioxidant capacity of plasma was significantly reversed in AFMK pretreated mice. Moreover, AFMK showed a very high level of in vitro hydroxyl radical scavenging potential which was measured by an electron spin resonance (ESR) study of the 2-hydroxy-5,5-dimethyl-1-pyrrolineN-oxide (DMPO-OH) adduct. IC50 values resulting from ESR analysis was 338.08 nm. The present study indicate that AFMK is a potent antioxidant in both in vivo and in vitro systems.