Reduced estrogen levels occurring during menopause in women are accompanied by a variety of disorders, e.g. hot flushes, depressions, osteoporosis, increase in body weight and reduced movement drive. The phytoestrogen genistein (GEN) has been demonstrated to have a significant bone-protective potency. In order to study the ER subtype-specific effects of this phytoestrogen on bone in an animal model, ovariectomized (OVX) female Wistar rats were either treated with 17beta-estradiol (E(2)) (4 microg/kg/day), the ER alpha-specific agonist (ALPHA) 16 alpha-LE(2) (10 microg/kg/day), the ER beta-specific agonist (BETA) 8 beta-VE(2) (100 microg/kg/day) or GEN (10 mg/kg/day) for 3 weeks. Vehicle-treated OVX animals served as controls. All animals had the opportunity of voluntary wheel running. Movement activity, changes of body weight and trabecular bone mineral density (BMD) in the tibia were analyzed. E(2) and ALPHA treatment, but not treatment with BETA, significantly increased the movement activity of OVX rats. Treatment with GEN resulted in a significant decrease of movement activity as compared to OVX animals. Bone mineral density in the trabecular area of the tibia and the expression of bone morphogenetic protein-2 (BMP-2) were significantly reduced in OVX- and BETA-treated rats as compared to rats substituted with E(2), ALPHA and GEN. The bone-protective effect of ALPHA was antagonized by co-treatment with the pure antiestrogen Faslodex (ICI). In order to distinguish hormone-dependent effects from those of exercise, we performed an additional experiment where the animals had no opportunity of wheel running. The results demonstrate that physically inactive rats have a stronger decrease of bone mineral density than physically active animals. Very surprisingly, our data demonstrate that GEN has no bone-protective activity in the absence of physical activity. In contrast, ALPHA and E(2) are bone-protective in the presence and absence of physical activity. In conclusion, our data provide evidence that the effects of E(2) on body weight, movement drive and protection of bone mineral density are mediated via ER alpha, whereas activation of ER beta has only a limited effect. Our data also indicate that the bone-protective effects of GEN may be mediated via ER alpha-dependent mechanisms and that physical activity has a strong impact on the bone-protective potency of this phytoestrogen.