A sensitive sandwich enzyme-linked immunosorbent assay (ELISA) for the mouse RANKL-OPG complex was developed by utilizing a polyclonal antibody that recognizes mouse soluble RANKL as an immobilized capture component and mouse OPG IgG labeled with peroxidase. We could quantify the RANKL-OPG complex level (detection limit: 1 pmol/L). Employing this assay system, we demonstrated that the RANKL-OPG complex was constitutively present in the serum of OPG+/- mice, but not in that of OPG-/- or wild-type C57BL/6 J mice.