Lipopolysaccharide (LPS) is a potent activator of tumor necrosis factor-α (TNF-α) production by macrophages. LPS stimulates the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 and increases TNF-α mRNA and protein accumulation in RAW 264.7 murine macrophages. However, the role of ERK1/2 activation in mediating LPS-stimulated TNF-α production is not well understood. Inhibition of ERK1/2 activation with PD-98059 or overexpression of dominant negative ERK1/2 decreased LPS-induced TNF-α mRNA quantity. LPS rapidly increased early growth response factor (Egr)-1 binding to the TNF-α promoter; this response was blunted in cells treated with PD-98059 or transfected with dominant-negative ERK1/2. Using a chloramphenicol acetyltransferase reporter gene linked to the Egr-1 promoter, we show that LPS increased Egr-1 promoter activity via an ERK1/2-dependent mechanism. These results delineate the role of ERK1/2 activation of Egr-1 activity in mediating LPS-induced increases in TNF-α mRNA expression in macrophages.