Alcohol use is typically associated with impaired immunity and increased host susceptibility to infection, partially due to decreased inflammatory response. Acute ethanol exposure has been shown to down-regulate monocyte production of inflammatory cytokines. Activation of the pluripotent transcription factor NFkappaB is a pivotal step in the induction of inflammatory cytokines, chemokines and growth factors. Therefore, we hypothesized that alcohol may alter NFkappaB activation, thus providing a mechanism for the decreased inflammatory cytokine production by monocytes after acute alcohol treatment. We show here for the first time that alcohol inhibits lipopolysaccharide (LPS)-induced NFkappaB activation in human monocytes by decreasing DNA binding of the p65/p50 heterodimer as seen in electrophoretic mobility shift and supershift assays. We also demonstrate that alcohol prevents LPS-induced nuclear translocation of p65 and to a lesser extent that of the p50 subunits. NFkappaB activation is regulated via phosphorylation and proteolytic degradation of IkappaB. Thus, we investigated the effect of acute ethanol treatment on IkappaB in human monocytes. Alcohol did not prevent LPS-induced IkappaBalpha degradation but decreased the levels of phospho-specific IkappaBalpha (Ser32). Finally, for the first time we show that de novo protein synthesis is necessary to bring about the ethanol-mediated inhibition of LPS-induced NFkappaB activation. Consequently, these results suggest that physiologically relevant concentrations of alcohol interfere with NFkappaB activation and thereby may affect the regulation of NFkappaB-controlled gene activation.