Nuclear factor kappa B (NF-kappa B) is an inducible transcription factor that positively regulates the expression of proimmune and proinflammatory genes, while glucocorticoids are potent suppressors of immune and inflammatory responses. NF-kappa B and the glucocorticoid receptor (GR) physically interact, resulting in repression of NF-kappa B transactivation. In transient cotransfection experiments, we demonstrate a dose-dependent, mutual antagonism between NF-kappa B and GR. Functional dissection of the NF-kappa B p50 and p65 subunits and deletion mutants of GR indicate that the GR antagonism is specific to the p65 subunit of NF-kappa B heterodimer, whereas multiple domains of GR are essential to repress p65-mediated transactivation. Despite its repression of GR transactivation, p65 failed to block the transrepressive GR homologous down-regulation function. We also demonstrate that negative interactions between p65 and GR are not selective for GR, but also occur between NF-kappa B and androgen, progesterone B, and estrogen receptors. However, although each of these members of the steroid hormone receptor family is repressed by NF-kappa B, only GR effectively inhibits p65 transactivation. Further, in cotransfections using a chimeric estrogen-GR, the presence of the GR DNA-binding domain is insufficient to confer mutual antagonism to the p65-estrogen receptor interaction. Selectivity of p65 repression for each steroid receptor is demonstrated by I kappa B rescue from NF-kappa B-mediated inhibition. Together these data suggest that NF-kappa B p65 physically interacts with multiple steroid hormone receptors, and this interaction is sufficient to transrepress each steroid receptor. Further, the NF-kappa B status of a cell has the potential to significantly alter multiple steroid signaling pathways within that cell.