The product of melatonin oxidation, N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK), was synthesized and a method for its determination in biological samples was developed. High performance liquid chromatography (HPLC) with fluorescence detection provided good sensitivity and selectivity. Wavelengths of 350 and 480 nm were used for excitation and emission, respectively. Serum and retinal homogenates were extracted with chloroform prior to analysis by HPLC. Endogenous AFMK was detected in the retina of rats but the serum concentration of this melatonin metabolite was below the detection limit of the method for measurement. Retinal AFMK concentration was higher during the dark phase of the light/dark cycle, when the retinal melatonin content is maximal. Intraperitoneal administration of melatonin significantly increased serum and retinal AFMK levels. Formation of AFMK from melatonin was also confirmed by in vivo microdialysis with the probe implanted into the brain lateral ventricle. The study shows that AFMK is indeed a product of melatonin oxidation in vivo. The possible physiological significance of melatonin oxidation metabolic pathway is discussed.