In order to characterize the regulation of the gene encoding the p50 subunit of the transcription factor NF-kappa B, we have isolated a human genomic clone, and sequenced the promoter of this gene. By in situ hybridization we have mapped the gene encoding the p50 subunit of NF-kappa B to the 4q23-4q25 region of the human genome and the H1-H3 region of the murine chromosome 3. The p50 promoter lacks TATA and CAAT elements, but contains NF-kappa B, AP-1 and HIP-1 binding sequence. The kappa B motif binds NF-kappa B, KBF1, and heterodimers of p50 and c-rel, suggesting that the gene is regulated by members of this family. Co-transfection experiments demonstrate that the p50 promoter can be activated by either of the two subunits of NF-kappa B (p50 and p65), and more strongly by the combination of both. The promoter of p50 can be activated by phorbol esters and tumor necrosis factor alpha but not by forskolin and these responses are mediated through the NF-kappa B binding sequence. The involvement of NF-kappa B in the regulation of the p50 gene is also supported by the inhibition of the PMA activation of the promoter by an NF-kappa B transdominant negative mutant, as well as the product of the v-rel oncogene.