Expression of the manganese superoxide dismutase (Mn-SOD) is induced by pro-inflammatory cytokines. We investigated the cis-acting elements within a tumor necrosis factor-responsive element (TNFRE) which was identified in the second intron of the murine Mn-SOD gene. Site-directed mutagenesis, reporter plasmid transfection studies and electrophoretic mobility shift assays demonstrated that inducible transcription factors enhanced the transcriptional activity of the Mn-SOD gene through the TNFRE. The cooperation between proteins binding to the newly identified NF-kappaB and C/EBP sites led to synergistic gene transcription. This report provides the first evidence that cooperation between two distinct cis-acting elements may be required for induction of Mn-SOD gene expression mediated by lipopolysaccharide and interferon-gamma.