The livers of alcohol fed animals seem to be conditioned to be more susceptible to a variety of second hits. A variety of evidence has suggested that the second hit may be mediated by the innate immune response, including cytokines. Ethanol promotes lipopolysaccharide (LPS) exposure via gut permeabilization, primes Kupffer cells to respond more strongly to LPS, and/or sensitizes target cells to the pro-inflammatory or lethal actions of cytokines. Gustot et al. examined the effect of oral ethanol feeding in mice on one aspect of this putative cascade, namely the expression and action of toll-like receptors (TLR) 1-9, the gatekeepers of the innate immune response. Previous alcohol work of others examined only the role of TLR4, the LPS receptor, but did not consider the other TLRs which respond to other bacterial and viral molecules. After 10 days, the oral ethanol feeding induced hepatic steatosis but no inflammation. Real-time PCR identified substantial increases in mRNA for TLR1,2,4,6,7,8,9 but not TLR 3 and 5 in response to ethanol. The mice were then challenged with ligands specific for individual receptors and exhibited increased TNF mRNA only in response to the ligands for receptors which were upregulated by ethanol, accompanied by marked increase in ALT and liver inflammatory foci. Antibiotics decreased fecal flora and moderately but significantly attenuated the hepatic steatosis (27.5% decrease) but did not decrease the effect of ethanol on expression of TLRs. Alcohol feeding was accompanied by increased lipid peroxidation and decreased GSH. Inhibition of NADPH oxidase with DPI suppressed oxidative stress and attenuated the ethanol induced steatosis while reducing the upregulation of TLR 2,4,6, and 9 but not TLR 1,7, and 8. The study demonstrates that ethanol upregulates a repertoire of TLRs which sensitize the liver to injury induced by specific ligands for these receptors. Bacterial products including, but not limited to LPS, and viral molecules thus evoke a myriad of injurious responses through their specific TLR targets.