The spin-trapping method with 5,5-dimethyl-1-pyrroline Noxide (DMPO) has been widely accepted as an assay method to measure hydroxyl radical formation, and to detect the hydroxyl radical scavenging activity of a compound. Although unstability of DMPO is claimed because it turns yellow with time even at -20˚C in a sealed tube under vacuum, 1 the basic chemistry of DMPO and DMPO-OH has not been well understood.In a previous work, 2 we noticed very weak DMPO-OH signals in the baseline of a negative control spectrum which was obtained after standing DMPO solution at 37˚C for 24 h. We suspected that in a DMPO aqueous solution at a higher concentration and at a higher temperature, an appropriate amount of DMPO-OH might generate. And we considered that this reaction should provide a practical preparation method to obtain DMPO-OH radical as a simple aqueous solution which contains no other materials except unreacted DMPO. Also this DMPO-OH aqueous solution may be utilized in the investigation of the chemical or physicochemical properties of DMPO-OH radicals.When DMPO was dissolved in purified water and the solution was heated at 70˚C for 30 min, a sufficient amount of DMPO-OH radical formation was observed by ESR ( Fig. 1). Under an argon atmosphere, DMPO-OH formation was minimized to 1/4, and this slightly increased under a dioxygen atmosphere. This indicates that dioxygen participates in the reaction. Previously, Makino et al. observed DMPO-OH formation in an aqueous mixture of DMPO and 1 mM FeCl3. Based on this observation of the formation of iron chelate with DMPO at 77 K, and DMPO-OCH3 formation in the presence of CH3OH, researchers elucidated that nucleophilic attachment of water to DMPO should occur in the presence of Fe 3+ ion. 3 To study the effect of a small number of metal ions in purified water, we replaced the purified water with ultra-pure water for ICP-MS measurements; the latter should contain iron of no more than 1 ppb. In ultrapure water, and under an argon atmosphere, DMPO-OH signal was still observed after heating, although its intensity decreased to about 1/20. In this communication, we describe DMPO-OH radical formation in hot water, in which dissolved dioxygen participates in the reaction and metal ions such as Fe 3+ might catalyze DMPO-OH formation. University, Japan *3 Tokyo Metropolitan Industrial Technology Research Institute, Itabashi, Japan *4 Faculty of Pharmaceutical Sciences, Nagoya City University, Mizuho, Japan When an aqueous solution of 5,5-dimethyl-1-pyrroline N-oxide (DMPO) was heated at 70˚C for 30 min, formation of DMPO-OH was observed by ESR. This DMPO-OH radical formation was suppressed under an argon atmosphere. When water was replaced with ultra-pure water for ICP-MS experiments, DMPO-OH radical formation was also diminished. Under an argon atmosphere in ultra-pure water, the intensity of the DMPO-OH signal decreased to about 1/20 of that observed under aerobic conditions with regular purified water. The addition of hydroxyl radical scavengers such as mannitol did not a...