Pretreatment with 1nM 1,25-dihydroxyvitamin D 3 (1,25), or non-hypercalcemic Vitamin D analogs, upregulated the response of creatine kinase (CK) to 17β-estradiol (30nM E 2 ), raloxifene (3000nM RAL) or dihydrotestosterone (300nM DHT) in primary human bone cells. Previously, we reported that these osteoblast-like cells responded to gonadal steroids in a sex specific manner. Bone cells derived from pre-menopausal women showed greater stimulation of CK specific activity by E 2 than bone cells from post-menopausal women; in male-derived cells no age related difference was found. In this study, we treated cells derived from female or male bones, at different ages, with the side chain modified analogs of Vitamin D: CB 1093 (CB), EB 1089 (EB), MC 1288 (MC) and the demonstrably non-calcemic hybrid analog JK 1624 F2-2 (JKF), by daily addition of 1nM, for 3 days. On day 4, cells were incubated with sex steroids for 4h and cell extracts were prepared. Pretreatment with JKF or CB significantly upregulated the response to 30nM E 2 in all female-derived cells and to 300nM DHT in mature male-derived cells. In cells from older males, only JKF caused augmentation of DHT action. Bone cells from pre- or post-menopausal females responded to 3000nM RAL by increased CK activity to the same extent as to 30nM E 2 ; however, RAL and E 2 , when applied together, resulted in mutual annihilation of their agonist activities. Vitamin D analogs prevented the antagonistic effect of RAL in the presence of E 2 , possibly due to increased numbers of ERs. Both estrogen receptors, α (ERα) and β (ERβ), were expressed in male- as well as in female-derived cells. However, only in female-derived cells were ERα and ERβ upregulated by pretreatment with Vitamin D analogs. This study raises the possibility of testing combined Vitamin D analog and estrogen replacement treatment for post-menopausal women to prevent osteoporosis.