BACKGROUND To date, the potential effects of free radicals on the activities of intestinal brush-border membrane enzymes have received little attention. Therefore, we conducted a series of experiments to examine the effects of free radicals on various enzymatic activities of rat small intestinal brush-border membranes. METHODS An in vitro Fe2+/ascorbate oxygen-radical generating system and rat small intestinal brush-border membranes were used for this purpose. The rate of lipid peroxidation was used as an index of free radical damage. In addition, fluorescence polarization, fatty acid analyses, membrane delipidation techniques, and studies with antioxidants and metal cofactors were used. RESULTS Increased free radical formation was associated with the inhibiton of alkaline phosphatase activity, with no change in the activities of sucrase, maltase, leucine aminopeptidase, and gamma-glutamyl transpeptidase. Generation of free radicals also markedly decreased membrane lipid fluidity and altered fatty acid composition. Catalase, reduced glutathione, or alpha-tocopherol treatment reduced lipid peroxidation as well as inactivation of enzyme activity. The use of artificial fluidizers, Arrhenius plots, and membrane delipidation studies failed to show a relationship between the inactivation of this enzyme and changes in membrane lipid composition or fluidity. CONCLUSIONS The inactivation of alkaline phosphatase by free radicals appears to involve its direct oxidation.