Role of glutathione peroxidase in iron-thiol-mediated lipid peroxidation was examined. The enzyme was unable to prevent peroxidation of extracted rat liver microsomal lipids. In contrast, when arachidonic acid was the substrate, glutathione peroxidase did decrease the formation of thiobarbituric acid-reactive material. Superoxide dismutase produced a consistent but partial inhibition of peroxidation and catalase was without effect. Our results suggest that iron-thiol-dependent lipid peroxidation cannot be completely blocked by protective enzymes that are effective in other systems.