The orphan nuclear receptors BmE75 and BmHR3 are induced by 20-hydroxyecdysone in the ovary of the silk moth Bombyx mori at the beginning of pupation and show stage-specific expression in ovarian follicles during pharate adult development. To analyze the function of these receptors, we have developed a transactivation assay based on the transcriptional stimulation of a retinoic acid receptor-related receptor response element (RORE)-linked promoter-reporter construct. Co-transfection of a Bombyx cell line with a BmHR3A expression construct results in constitutive activation of the reporter, whereas expression of BmE75 has no measurable effects on reporter expression. However, when the BmE75 receptors are co-introduced with BmHR3A into the cells, the BmHR3A-mediated transactivation is repressed. Repression of BmHR3A by BmE75 occurs by two distinct mechanisms. Increasing doses of BmE75 efficiently displace BmHR3A bound to the RORE target site in gel retardation assays, indicating that both receptors compete for common DNA target sites. However, analysis of the function of deletion mutants of BmE75 in the transactivation assay indicates that repression can also occur in the absence of the DNA-binding domain and that the C-terminal F domain is sufficient for repression. In gel retardation assays, the two receptor types form a ternary complex on a single RORE, suggesting that repression is also mediated by protein interactions on the DNA target site. Yeast two-hybrid assays show that BmHR3A interacts with BmE75 and that this interaction is dependent on the C terminus of BmHR3A and the F domain of BmE75. Because the C terminus of BmHR3A contains a strong activation domain, we predict that BmE75 blocks activation by BmHR3A through competition for co-activator binding sites located at the C terminus of BmHR3A. Our data also indicate that the transcriptional activities of BmHR3A and BmE75 are integrated in such a way that activation of RORE-linked target genes depends on the relative expression levels of the two receptor types.