TNF-alpha transcriptionally regulates murine monocyte chemoattractant protein-1 (MCP-1) expression. Three approaches were used to determine the mechanism by which TNF regulates MCP-1. Mutation analysis showed that two distal kappa B sites, a novel dimethylsulfate-hypersensitive sequence, and a promoter proximal SP-1 site were required for TNF induction. Although the kappa B sites and the hypersensitive sequence function as a NF-kappa B-mediated enhancer, regulating induction by TNF, stereospecific alignment of the kappa B sites was not critical. Trans-activation studies conducted by cotransfection of p50 and/or p65 expression vectors with MCP-1 constructions showed that TNF regulates MCP-1 through NF-kappa B. Examination of MCP-1 induction in NF-kappa B-disrupted embryonic fibroblasts showed that p65 was necessary for both the induction and the TNF-induced protein occupancy of the enhancer in vivo. The action of the antioxidant inhibitor of NF-kappa B activation, pyrrolidine dithiocarbamate, in wild-type and NF-kappa B mutant cells was examined. The results suggested that TNF activates NF-kappa B through both pyrrolidine dithiocarbamate-sensitive and -insensitive mechanisms. This study illustrates the crucial role for NF-kappa B p65 in the induction of the MCP-1 gene by TNF and in the assembly of a NF-kappa B dependent enhancer in vivo.