Summary Auto-oxidation of 5,6,7,8-tetrahydroneopterin (NPH 4 ) in phosphate buffer solution (PBS) was examined using reversed-phase high-performance liquid chromatography. In the chromatograms obtained every 30 min after NPH 4 was dissolved in PBS, the peak of NPH 4 gradually decreased and almost disappeared 90 min after the dissolution. In contrast, another peak of an unidentified substance (one of dihydroneopterins), appeared from the first time, gradually increased and became most dominant among various peaks 60 min after. The peak of 7,8-dihydroneopterin (7,8-NPH 2 ) appeared 30 min after and gradually increased, but its height was less than that of the above-mentioned peak from first to last. These results indicate that NPH 4 is readily oxidized by dissolved oxygen in PBS to produce 7,8-NPH 2 and other dihydroneopterins before the reaction with oxygen free radicals and that the substances which actually serve as antioxidants in vivo are these dihydroneopterins.