Retinoid receptor-related testis-associated receptor (RTR)/germ cell nuclear factor is a nuclear orphan receptor that plays an important role in the control of gene expression during early embryonic development and gametogenesis. It has been shown to repress transcriptional activation. In this study, we further characterize this repressor function. We demonstrate that RTR can suppress the transcriptional activation induced by the estrogen receptor related-receptor α1 through its response element. The latter is at least in part due to competition for binding to the same response element. In addition, RTR inhibits basal transcriptional activation, indicating that it functions as an active repressor. Mammalian two-hybrid analyses showed that RTR interacts with the co-repressor nuclear co-repressor (N-CoR) but is unable to interact with the co-repressor SMRT or RIP140. Pull-down analyses with glutathione S-transferase-RTR fusion protein demonstrated that RTR physically interacts with N-CoR in vitro, suggesting a potential role for N-CoR in the transcriptional repression by RTR. To identify the regions in RTR essential for the binding of RTR to N-CoR, the effect of various deletion and point mutations on this interaction was examined. This analysis revealed that this interaction requires the hinge domain, helix 3 as well as the helix 12 region of RTR. The residues Ser246-Tyr247 in the hinge domain, Lys318 in helix 3, and Lys489-Thr490 in helix 12 are identified as being critical in this interaction. Our results demonstrate that RTR can function as an active transcriptional repressor and that this repression can be mediated through interactions with the co-repressor N-CoR. We show that this interaction exhibits several characteristics unique to RTR. Through its repressor function, RTR can suppress the induction of transcriptional activation by other nuclear receptors. These repressor activities may provide important mechanisms by which RTR regulates gene expression during development and gametogenesis.