Expression of the manganese superoxide dismutase (Mn-SOD) is induced by tumor necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1), and lipopolysaccharide (LPS). Recently, a TNF-responsive element (TNFRE) was identified within the second intron of the murine Mn-SOD gene. The 5' CCAAT/enhancer binding protein (C/EBP)-related region within the TNFRE was responsive to TNF, whereas the 3' NF-kappaB-related region alone was not. This report describes the minimal promoter region of the Mn-SOD gene and investigates the cis-acting elements and trans-acting factors responsible for TNF-alpha-induced Mn-SOD gene expression. Reporter plasmid transfection studies demonstrated that inducible transcription factors enhanced the transcriptional activity of the Mn-SOD gene through the intronic enhancer region. Electrophoretic mobility shift assays demonstrated that after TNF-alpha stimulation, p50 and p65 NF-kappaB subunits bound specifically to the newly identified NF-kappaB transcription factor-binding site, distinct from the previously described NF-kappaB site, within the intronic enhancer region. In addition, site-directed mutagenesis and cotransfection studies demonstrated that the NF-kappaB p65 subunit enhanced the transcriptional activity of the Mn-SOD gene through the newly identified NF-kappaB site. These results show that a NF-kappaB p65 subunit is mainly involved in the molecular mechanisms controlling TNF-alpha-mediated Mn-SOD gene transcription.