Glucocorticoids inhibit inflammation by acting through the glucocorticoid receptor (GR) and powerfully repressing nuclear factor-kB (NF-kB) function (1). Ligand binding to the C-terminal of GR promotes GR nuclear translocation and binding to NF-kB through the GR DNA binding domain. NFkB is a multiprotein family of transcription factors, activated by proinflammatory cytokines. Family members all contain a conserved Rel domain, which binds DNA. Importantly, transcriptome profiling experiments have shown that expression of the p65 component of NFkB predicts the glucocorticoid response of bronchial asthma, a major human inflammatory disease widely treated with synthetic glucocorticoid drugs (2). Expression of NFkB p65 in cells impairs GR function. To identify how GR ligand binding influences the functional interaction between NF-kB and GR, studies were performed in human cell line models. Both dexamethasone (agonist), and RU486 (antagonist) promote efficient nuclear translocation of the GR, and we show occupancy of the same intranuclear compartment as NF-kB with both ligands. However, unlike dexamethasone, RU486 had negligible activity to inhibit NF-kB transactivation. This failure may stem from altered co-factor recruitment, or altered interaction between the GR and NF-kB. Using a GST pull-down approach we found that RU486 appeared to disrupt the GR-NFkB interaction. Following this bioluminescence resonance energy transfer (BRET) analysis identified a major glucocorticoid ligand effect on interaction between the GR and the p65 component of NF-kB, with RU486 inhibiting recruitment compared with dexamethasone. Using the BRET assay, we found that RU486 efficiently recruited NCoR to the GR, unlike dexamethasone, which recruited SRC1. Therefore RU486 promotes differential protein recruitment to both the C terminal (NCoR and SRC1) and DNA binding domain of the receptor (NFkB). By disrupting the GR N-terminal, which contains the major transactivation domain AF-1, we were able to unmask significant transrepressive activity of RU486 on NFkB. Chromatin immunoprecipitation studies showed efficient recruitment of NFkB p65 to the NFkB response element on the interleukin 8 gene in response to TNF treatment, as expected. Also as expected, glucocorticoid treatment promoted recruitment of GR to the same element, but only under conditions that allowed prior recruitment of NFkB. Importantly, we show that impaired interaction between GR and p65 with RU486 leads to reduced recruitment of the GR to the NF-kB responsive region of the IL-8 promoter, again in contrast to dexamethasone (1). We demonstrate that ligand-induced conformation of the GR C-terminal has profound effects on the functional surface generated by the DNA binding domain of the GR. This has implications for understanding ligand-dependent interdomain communication.