Enzymatically generated tyrosyl radicals are effectively scavenged by reduced glutathione (GSH), thereby generating glutathione thiyl radicals and superoxide radicals, subsequently. Here, we have used horseradish peroxidase to generate tyrosyl radicals and investigated the fate of the superoxide radicals. At low GSH concentrations (with a maximum effect at 250 microM) a major reaction was between superoxide and the phenoxyl radical leading to a tyrosine peroxide. Formation of the peroxide was confirmed using a peroxide-specific colorimetric assay and detection of a new HPLC-peak. Its formation was inhibited by superoxide dismutase (SOD). The peroxide decomposed slowly in a reaction that was accelerated by GSH to give a new chromatographic peak. Increasing the GSH concentration decreased the amount of tyrosine peroxide formed and caused increases in rates of oxygen uptake and GSH oxidation. These increases were not seen in the presence of SOD and are consistent with GSH scavenging superoxide and leading to oxygen-dependent chain oxidation of GSH. Both pathways are undesirable for the cell and are effectively suppressed only if GSH as a radical scavenger acts in concert with SOD.