Recent development in the field of gene regulation by nuclear receptors (NRs) have identified a role for cofactors in transcriptional control. While some of the NR-associated proteins serve as coactivators, the effect of the receptor interacting protein 140 (RIP140) on NR transcriptional responses is complex. In this report we have studied the effect of RIP140 on gene regulation by the glucocorticoid receptor (GR). We demonstrate that RIP140 antagonized all GR-mediated responses tested, which included activation through classical GRE, the synergistic effects of glucocorticoids on AP-1 and Pbx1/HOXB1 responsive elements, as well as gene repression through a negative GRE and cross-talk with NF-kappaB (RelA). This involved the ligand-binding domain of the GR and did not occur when the GR was bound to the antagonist RU486. The strong repressive effect of RIP140 was restricted to glucocorticoid-mediated responses in as much as it slightly increased signaling through the RelA and the Pit-1/Pbx proteins and only slightly repressed signaling through the Pbx1/HOXB1 and AP-1 proteins, excluding general squelching as a mechanism. Instead, this suggests that RIP140 acts as a direct inhibitor of GR function. In line with a direct effect of RIP140 on the GR, we demonstrate a GR-RIP140 interaction in vitro by a glutathione S-transferase-pull down assay. Furthermore, the repressive effect of RIP140 could partially be overcome by overexpression of the coactivator TIF2, which involved a competition between TIF2 and RIP140 for binding to the GR.