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N-[4-H-3]Benzoylglycylglycylglycine ([H-3]BzG(3)) was tested as a probe for detecting hydroxyl radicals (*OH). Aerated solutions of L-ascorbate generated *OH, which oxidized [3 H]BzG3, yielding hydrophilic (probably hydroxylated) derivatives plus tritiated water. 3 The (H2O)-H-3 was separated from organic products and remaining [H-3]BzG(3) on Dowex-1. (H2O)-H-3 production was much greater with *OH than with other reactive oxygen species (ROS) (e.g., H2O2, superoxide). The alight (H2O)-H-3 production in the presence of H2O2 or superoxide was blocked by *OH scavengers (e.g., glycerol, mannitol, butan-1-ol) that do not scavenge H2O2 or superoxide. This indicates that (H2O)-H-3 production was caused by *OH and that other ROS only generated any (H2O)-H-3 by forming traces of *OH. Doses of *OH that caused detectable nonenzymic polysaccharide scission also caused (H2O)-H-3 production, indicating that [H-3]BzG(3) is a sensitive *OH probe in studies of polymer scission. The ability of scavengers and chelators to protect against ascorbate-mediated polysaccharide scission paralleled their ability to inhibit concurrent (H2O)-H-3 production, indicating that both processes were due to *OH. Thus, [3 H]BzG3 is a simple, specific, sensitive, and robust probe for detecting *OH production in vitro. It may have applications for in vivo detection of extracellular *OH in arthritic joints and of apoplastic *OH in plant cell walls. (C) 2004 Elsevier Inc. All rights reserved.