Tibolone (Org OD 14) is a synthetic steroid with combined estrogenic, progestagenic, and androgenic properties and behaves as a tissue-specific steroid. In the current study, we determined the effects of a 4-week treatment with different doses of tibolone on estrogen deficiency-induced bone loss in mature 3-month-old rats. As a reference, 17alpha-ethinyl estradiol (EE2) was used. The frequency of administration, once or twice a day, was also studied. Bone parameters were determined in sham operated controls, ovariectomized (OVX) controls and OVX-treated rats. Bone loss was assessed by peripheral quantitative computed tomography directly and by quantitative Roentgen densitometry after defatting to exclude influence of fat changes. Femoral bone geometric parameters, plasma osteocalcin level, and urinary deoxypyridinoline/creatinine ratio were also determined. Ovariectomy caused a significant decrease in trabecular bone mineral density in the distal metaphyseal part of the femur using both methods, whereas no change in cortical bone density was found. Trabecular bone loss was prevented in a dose-dependent manner by tibolone (250, 1000, and 4000 microgram/rat/day) when given once or twice daily. EE2 also prevented trabecular bone loss but its efficacy was dependent upon the frequency of dosing. Both tibolone and EE2 induced a significant reduction in the urinary deoxypyridinoline/creatinine ratio and plasma osteocalcin level. Tibolone and EE2 had no effect on other femoral bone parameters except a reduction in femoral length. In conclusion, treatment with tibolone for 4 weeks prevented OVX-induced bone loss by suppressing both bone resorption and bone turnover in a similar way as EE2. However, the frequency of dosing is more important for EE2 than for tibolone. Tibolone acts in this animal model for postmenopausal bone loss as an estrogen agonist on bone.