Acrolein is a highly electrophilic ␣,␤-unsaturated aldehyde to which humans are exposed in various situations. In the present study, the effects of sublethal doses of acrolein on nuclear factor B (NF-B) activation in A549 human lung adenocarcinoma cells were investigated. Immediately following a 30-min exposure to 45 fmol of acrolein/cell, glutathione (GSH) and DNA synthesis and NF-B binding were reduced by more than 80%. All parameters returned to normal or supranormal levels by 8 h post-treatment. Pretreatment with acrolein completely blocked 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced activation of NF-B. Cells treated for 1 h with 1 mM diethyl maleate (DEM) showed a 34 and 53% decrease in GSH and DNA synthesis, respectively. DEM also reduced NF-B activation by 64% at 2 h posttreatment, with recovery to within 22% of control at 8 h. Both acrolein and DEM decreased NF-B function ϳ50% at 2 h after treatment with TPA, as shown by a secreted alkaline phosphatase reporter assay. GSH returned to control levels by 8 h after DEM treatment, but proliferation remained significantly depressed for 24 h. Interestingly, DEM caused a profound decrease in NF-B binding, even at doses as low as 0.125 mM that had little effect on GSH. Neither acrolein nor DEM had any effect on the levels of phosphorylated or nonphosphorylated inhibitor B-␣ (IB-␣). Furthermore, acrolein decreased NF-B activation in cells depleted of IB-␣ by TPA stimulation in the presence of cycloheximide, demonstrating that the decrease in NF-B activation was not the result of increased binding by the inhibitory protein. This conclusion was further supported by the finding that acrolein modified NF-B in the cytosol prior to chemical dissociation from IB with detergent. Together, these data support the conclusion that the inhibition of NF-B activation by acrolein and DEM is IB-independent. The mechanism appears to be related to direct modification of thiol groups in the NF-B subunits.