Toll-like receptors (TLRs) play a fundamental role in the immune system by detecting pathogen associated molecular patterns (PAMPs) to sense host infection. Ethanol at doses relevant for humans inhibits the pathogen induced cytokine response mediated through TLRs. The current study was designed to investigate the mechanisms of this effect by determining whether ethanol inhibits TLR3 and TLR4 mediated TNF- secretion through inhibition of transcription factor activation or post-transcriptional effects. In NF-B reporter mice, activation of NF-B in vivo by LPS was inhibited by ethanol (LPS alone yielded 170,00035,300 arbitrary units of light emission; LPS plus ethanol yielded 56,12016880, p=0.04). Inhibition of protein synthesis by cycloheximide revealed that poly I:C- or LPS-induced secreted TNF- is synthesized de novo, not released from cellular stores. Using real time RT-PCR, we found inhibition of LPS and poly I:C induced TNF- gene transcription by ethanol. Using an inhibitor of tumor necrosis factor alpha converting enzyme (TACE), we found that shedding caused by TACE is a prerequisite for TNF- release after pathogen challenge. Flow cytometry was used to investigate if ethanol decreases TNF- secretion by inhibition of TACE. In cells treated with LPS, ethanol decreased both TNF- cell surface expression and secretion. For example, 4.690.60% of untreated cells were positive for cell surface TNF-, LPS increased this to 25.180.85%, which was inhibited by ethanol (86.8 mM) to 14.290.39% and increased by a TACE inhibitor to 57.880.62%. In contrast, cells treated with poly I:C had decreased secretion of TNF- but not cell surface expression. There was some evidence for inhibition of TACE by ethanol in the case of LPS, but decreased TNF- gene expression seems to be the major mechanism of ethanol action in this system.