Oxidative stress frequently leads to altered function of membrane proteins. Isoketals are highly reactive products of the isoprostane pathway of free radical-induced lipid peroxidation that rapidly form covalent protein adducts and exhibit a remarkable proclivity to form protein cross links in vitro. Examination of isoketal adducts from an animal model of oxidative injury revealed that initial adducts were formed by isoketals esterified in phospholipids, representing a novel oxidative injury-associated modification of proteins by phospholipids. Maturation of adducts involved cleavage from phospholipids and conversion of adducts to a more stable chemical form that can be detected for extended periods. Because initial adducts were formed by phospholipid-esterified isoketals, the functional consequence of isoketal adduction was examined using a model membrane protein (a cardiac K(+) channel). These studies revealed that isoketal adduction profoundly altered protein function, inhibiting potassium current in a dose-dependent manner. These findings indicate that phospholipid-esterified isoketals rapidly adduct membrane proteins and that such modification can alter protein function, suggesting a generalized cellular mechanism for alteration of membrane function as a consequence of oxidative stress.