A unique heterodimerization pathway involving orphan receptors TR2 and TR4 is demonstrated. TR2 and TR4 preferentially form heterodimers in solution as well as on DNA elements containing a direct repeat-5 (DR5). The in vitro interaction between TR2 and TR4 is demonstrated by the yeast and the mammalian two-hybrid interaction assays, the pull-down assay, and the gel mobility shift assay. The in vivo interaction is demonstrated by following the intracellular localization of fusion receptors tagged with a green fluorescent protein. The dimerization is mediated by the ligand binding domains, and the three leucine residues on helix 10 of TR2 are critical for this interaction. In addition, coexpression of these two receptors exerts a much stronger repressive activity on a DR5-containing reporter than expressing either receptor alone. In the developing testis, TR2 and TR4 are coexpressed in the same testicular cell populations and exhibit a parallel pattern of expression along development. The preferential heterodimerization between TR2 and R4 and their coexistence in specific germ cell populations suggest a physiological role of TR2/TR4 heterodimers in germ cell development.