Receptor activator of nuclear factor κB (RANK), its ligand RANKL and the RANKL decoy receptor osteoprotegerin (OPG) form a protein triad of importance for bone homeostasis maintenance. Estradiol-17ß (E2), testosterone (T) and the Cimicifuga racemosa extract BNO 1055 (CR BNO 1055) have antiosteoporotic effects following gonadectomy of female or male rats. Their mechanisms of action, however, remain unexplored. Rats were ovariectomized (ovx) or orchidectomized (orx) and substituted with E2, T and CR BNO 1055 via soy free food for 3 months. Cancellous bone mineral density of the tibia metaphysis was determined by quantitative computer tomography. Serum levels of RANKL, OPG, osteocalcin and RatLaps were determined by radioimmunoassays. In both, ovx and orx animals RANKL was suppressed by E2 and CR BNO 1055, while T suppressed RANKL levels in orx males only. OPG remained uneffected. In both sexes, osteocalcin was significantly reduced by E2. RatLaps were reduced by E2 and CR BNO 1055. E2 treated animals had the highest bone mineral density; this effect was partly shared by CR BNO 1055, but not by T. Thus, the bone sparing effect of E2 and CR BNO 1055 are partly mediated by inhibition of RANKL production. The E2 effects are most likely mediated via the estrogen receptor alpha. Since compounds in CR BNO 1055 do not bind to any type of estrogen receptors, the mechanism of RANKL inhibition by CR BNO 1055 remains unknown. The failure of T to prevent osteoporosis in orx animals despite reduced RANKL serum concentrations remains enigmatic.