Increased production of tumor necrosis factor alpha (TNFalpha) is associated with the development of alcoholic liver disease. Culture of RAW264.7 macrophages with 25 mm ethanol for 48 h increased lipopolysaccharide (LPS)-stimulated accumulation of tumor necrosis factor alpha (TNFalpha) peptide and mRNA by 2-fold. We investigated whether chronic ethanol-induced increases in the DNA binding and/or promoter activity of the key transcription factors regulating LPS-stimulated TNFalpha promoter activity contribute to increased TNFalpha expression. Binding of Egr-1 to the TNFalpha promoter was increased by 2.5-fold after ethanol exposure, whereas NFkappaB binding was decreased to 30% of control. AP-1 binding was not affected. Changes in binding activity were paralleled by an increased contribution of the Egr-1 binding site and a decreased contribution of the NFkappaB site to LPS-stimulated TNFalpha promoter activity. Overexpression of dominant negative Egr-1 prevented the ethanol-induced increase in LPS-stimulated TNFalpha mRNA accumulation. Chronic ethanol exposure enhanced LPS-stimulated Egr-1 promoter-driven CAT expression and transcription of Egr-1. Induction of Egr-1 is dependent on ERK1/2 activation in other systems. Therefore, we investigated whether the ERK1/2 pathway mediated the chronic ethanol-induced increases in Egr-1 and TNFalpha. Increased Egr-1 promoter activity and TNFalpha mRNA accumulation after chronic ethanol were both prevented by overexpression of dominant negative ERK1/2. LPS-stimulated ERK1/2 phosphorylation was increased 2-fold in cells cultured with ethanol compared with controls. These results demonstrate that enhanced LPS-dependent activation of Egr-1 contributes to increased TNFalpha production after chronic ethanol exposure.