This report describes studies yielding additional evidence that superoxide anion (O2) production by some biological oxidoreductase systems is a potential source of hydroxyl radical production. The phenomenon appears to be an intrinsic property of certain enzyme systems which produce superoxide and H2O2, and can result in extensive oxidative degradation of membrane lipids. Earlier studies had suggested that iron (chelated to maintain solubility) augmented production of the hydroxyl radical in such systems according to the following reaction sequence: O2 + Fe3+ leads to O2 + Fe2+ Fe2+ + H2O2 leads to Fe3+ + HO-+OH-. The data reported below provide additional support for the occurrence of these reactions, especially the reduction of Fe3+ by superoxide. Because the conditions for such reactions appear to exist in animal tissues, the results indicate a mechanism for the initiation and promotion of peroxidative attacks on membrane lipids and also suggest that the role of antioxidants in intracellular metabolism may be to inhibit initiation of degradative reactions by the highly reactive radicals formed extraneously during metabolic activity. This report presents the following new information: (1) Fe3+ is reduced to Fe2+ during xanthine oxidase activity and a significant part of the reduction was oxygen dependent. (2) Mn2+ appears to function as an efficient superoxide anion scavenger, and this function can be inhibited by EDTA. (3) The O2-dependent reduction of Fe3+ to Fe2+ by xanthine oxidase activity is inhibited by Mn2+, which, in view of statement 2 above, is a further indication that the reduction of the iron involves superoxide anion. (4) Free radical scavengers prevent or reverse the Fe3+ inhibiton of cytochrome c3+ reduction by xanthine oxidase. (5) The inhibition of xanthine oxidase-catalyzed reduction of cyt c3+ by Fe3+ does not affect uric acid production by the xanthine oxidase system. (6) The reoxidation of reduced cyt c in the xanthine oxidase system is markedly enhanced by Fe3+ and is apparently due to enhanced HO-RADICAL formation since the Fe3+-stimulated reoxidation is inhibited by free radical scavengers, including those with specificity for the hydroxyl radical.