Recently, we have reported the cloning of the germ cell-specific, nuclear orphan receptor germ cell nuclear factor (GCNF)/RTR. In this study, we characterize the RTR response elements by an electrophoretic mobility shift assay/polymerase chain reaction-based, DNA binding site selection strategy. RTR binds with the greatest affinity to response elements containing TCA(AG(G/T)TCA)2 (consensus RTR response element; conRTRE), to which it binds as a homodimer. RTR is also able to bind as a monomer to a single core motif TCAAG(G/T)TCA, albeit with a lower affinity. Mutation analysis supports the specific requirements of the 5'-flanking sequence and the core motif of the RTRE for optimal binding of RTR. An RTR-specific antiserum (RTR-Ab2) was raised that causes supershift of the RTR-conRTRE complex in EMSA. Based on the sequence of the conRTRE, we located a putative RTRE, referred to as P2-RE, in the 5' promoter-flanking region of the mouse protamine 2 gene, which is induced during the same stage of spermatogenesis as RTR. The ability of RTR-Ab2 to cause a supershift of an RTR-RTRE complex with nuclear extracts from different tissues correlated with the tissue- and development-specific expression of RTR. Transfection of RTR in CV-1 cells was unable to cause RTRE-dependent transactivation of a CAT reporter gene; however, an RTR-VP16 fusion protein could induce transactivation through several RTREs, including P2-RE.