Technical Abstract: Acrolein is a highly electrophilic a,ß-unsaturated aldehyde present in a number of environmental sources, especially cigarette smoke. It reacts strongly with the thiol groups of cysteine residues by Michael addition and has been reported to inhibit nuclear factor-kB (NF-kB) activation by lipopolysaccharide (LPS). The mechanism by which it inhibits NF-kB is not clear. Toll-like receptors (TLRs) play a key role in sensing microbial components and inducing innate immune responses, and LPSinduced dimerization of TLR4 is required for activation of downstream signaling pathways. Thus, dimerization of TLR4 may be one of the first events involved in activating TLR4-mediated signaling pathways. Stimulation of TLR4 by LPS activates both myeloid differential factor 88 (MyD88)- and TIR domain-containing adapter inducing IFNß (TRIF)-dependent signaling pathways leading to activation of NF-kB and IFN-regulatory factor 3 (IRF3). Acrolein inhibited NF-kB and IRF3 activation by LPS, but it did not inhibit NF-kB or IRF3 activation by MyD88, inhibitor kB kinase (IKK)ß, TRIF, or TNFreceptor- associated factor family member-associated NF- kB activator (TANK)-binding kinase 1 (TBK1). Acrolein inhibited LPS-induced dimerization of TLR4, which resulted in the down-regulation of NF-kB and IRF3 activation. These results suggest that activation of TLRs and subsequent immune/inflammatory responses induced by endogenous molecules or chronic infection can be modulated by certain chemicals with a structural motif that enables Michael addition.