Hydralazine strongly suppresses the toxicity of acrolein, a reactive aldehyde that contributes to numerous health disorders. At least two mechanisms may underlie the cytoprotection, both of which involve the nucleophilic hydrazine possessed by hydralazine. Under the simplest scenario, hydralazine directly scavenges free acrolein, decreasing intracellular acrolein availability and thereby suppressing macromolecular adduction. In a second "adduct-trapping" mechanism, the drug forms hydrazones with acrolein-derived Michael adducts in cell proteins, preventing secondary reactions of adducted proteins that may trigger cell death. To identify the most important mechanism, we explored these two pathways in mouse hepatocytes poisoned with the acrolein precursor allyl alcohol. Intense concentration-dependent adduct- trapping in cell proteins accompanied the suppression of toxicity by hydralazine. However, protective concentrations of hydralazine did not alter extracellular free acrolein levels, cellular glutathione loss, or protein carbonylation, suggesting that the cytoprotection is not due to minimization of intracellular acrolein availability. To explore ways whereby adduct-trapping might confer cytoprotection, the effect of hydralazine on acrolein-induced protein crosslinking was examined. Using bovine pancreas ribonuclease A as a model protein, acrolein caused rapid time-and concentration-dependent cross-linking, with dimerized protein detectable within 45 min of commencing protein modification. Lysine adduction in monomeric protein preceded the appearance of oligomers, whereas reductive methylation of protein amine groups abolished both adduction and oligomerization. Hydralazine inhibited cross-linking if added 30 min after commencing acrolein exposure but was ineffective if added after a 90-min delay. Adduct-trapping closely accompanied the inhibition of cross-linking by hydralazine. These findings suggest that cross-link blocking may contribute to hydralazine cytoprotection.