trans-4-Oxo-2-nonenal (ONE) has recently been demonstrated to be a direct product of lipid peroxidation. In earlier studies to elucidate the structure of the trans-4-hydroxy-2-nonenal (HNE)-derived fluorescent Lys-Lys cross-link, we showed that ONE was capable of both oxidative and nonoxidative cross-linking of amines. A more comprehensive study on nonoxidative modification of protein nucleophiles by ONE is described here, focusing on the initial Michael addition of imidazole, thiol, and amine groups to C2 or C3 to give 4-keto aldehydes that can then condense with amines to form nucleophile-substituted pyrroles. 2,3-Substituted pyrroles (major) and 2,4-substituted pyrroles (minor) were distinguished by 2D NMR techniques, and N(tau)-substitution is preferred over N(pi)-substitution in the Michael addition of histidine. Mechanisms of both nonoxidative and oxidative side chain reactions of ONE are discussed, as is the relative propensity (ONE > HNE) to induce cross-linking of the model proteins ribonuclease A and beta-lactoglobulin.