The retinoid-related orphan receptor gamma (RORgamma) 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 RORgamma 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 RORgamma LBD. We demonstrate that the H3-4/H12 co-activator interaction surface of RORgamma displays a selectivity for LXXLL peptides that is distinct from those of other nuclear receptors. LXXLL peptides that interacted with RORgamma efficiently antagonized RORgamma-mediated transcriptional activation. Mutations E502Q and Y500F in H12, and K334A, Q347A, and I348D in H3 and H4 of RORgamma, 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 RORgamma(LBD) derived through homology modeling. These peptide antagonists provide a useful tool to analyze the conformation changes in the RORgamma(LBD) and to study RORgamma receptor signaling.