The modification of proteins by lipid aldehydes produced in cells undergoing oxidative stress has been proposed as an important event that contributes to the pathology of numerous diseases. In this context, the alpha,beta-unsaturated aldehydes 4-hydroxynonenal (4-HNE) and 4-oxononenal (4-ONE) generated during membrane lipid peroxidation have been shown to adduct and inactivate numerous proteins. We report here that purified bovine brain tubulin modified with physiologically relevant concentrations of 4-HNE or 4-ONE results in significant protein cross-linking and marked inhibition of the functional capacity of tubulin polymerization. Comparative analysis demonstrated that 4-ONE is a much more potent cross-linker and inhibitor of tubulin assembly than 4-HNE. Additional experiments revealed the unique property of 4-ONE, initiation of depolymerization of intact microtubules. LC-MS/MS analysis demonstrated that Cys 347alpha, Cys 376alpha, and Cys 303beta are consistently modified by 4-HNE. The identification of target residues within tubulin modified by 4-ONE was not successful, and this was attributed to the marked tubulin cross-linking that occurred immediately after addition of 4-ONE. The modification of Lys residues by reductive propylation demonstrated that the majority of 4-HNE and 4-ONE adducts involve Lys residues, suggesting that tubulin cross-links are Lys-dependent. Taken together, these data suggest a mechanistic basis for the impairment of tubulin function by 4-HNE and 4-ONE produced as a consequence of diseases associated with chronic oxidative stress.