To examine a potential role for phytoestrogens in postmenopausal bone loss, the oophorectomized (OOX) rat model has been used in three studies to investigate the effects of the phytoestrogens coumestrol, zearalanol and a mixture of isoflavones on estrogen-dependent bone loss. In the studies of coumestrol and zearalanol, the rats were allocated to a control group, a phytoestrogen-treated group (1.5 micromol coumestrol or 3.1 mmol zearalanol twice per week, intramuscular) or, in the coumestrol study, an estrogen-treated group (28.1 nmol, intramuscular). In the isoflavone study, the rats were allocated to a control group, an estrogen treated group or a treatment group that received 131.25 mg of phytoestrogens per week incorporated into the nonpurified rat diet. Bone mineral density was measured globally and at the spine and femur at base line and 6 wk post-oophorectomy. In the coumestrol study, blood and urine samples were collected. Compared with the control group, rats receiving coumestrol and zearalanol had significantly reduced bone loss at all sites measured. The estrogen-treated group had significantly greater bone density than the control and the coumestrol-treated groups in the spine and global measurements. Coumestrol reduced urine calcium excretion and the bone resorption markers pyridinoline and deoxypyridinoline after 1 wk of treatment. Oral isoflavone phytoestrogens had no effect on oophorectomized rats including bone loss at the dose used. Thus, for the first time, the bioactivity of coumestrol and zearalanol in preventing bone loss has been demonstrated in a well-recognized model of postmenopausal bone loss.