The retinoid-related orphan receptor γ (RORγ) has been shown to function as a positive regulator of transcription in many cell lines. Transcriptional activation by nuclear receptors involves recruitment of co-activators that interact with receptors through their LXXLL motifs (NR box). In this study, we analyze the interaction of RORγ with the co-activator SRC1 and use a series of LXXLL-containing peptides to probe for changes in the conformation of the co-activator interaction surface of the RORγ LBD. We demonstrate that the H3-4/H12 co-activator interaction surface of RORγ displays a selectivity for LXXLL peptides that is distinct from those of other nuclear receptors. LXXLL peptides that interacted with RORγ efficiently antagonized RORγ-mediated transcriptional activation. Mutations E 502Q and Y500F in H12, and K334A, Q 347A, and I348D in H3 and H4 of RORγ, severely impact the recruitment of LXXLL peptides. The effects of these mutations are consistent with predictions made on the basis of the structure of the RORγ(LBD) derived through homology modeling. These peptide antagonists provide a useful tool to analyze the conformation changes in the RORγ(LBD) and to study RORγ receptor signaling.