Rat and mice bones in vivo and their derived cell cultures in vitro respond sex-specifically to gonadal steroids (GS) by stimulating DNA synthesis (DNA) and the specific activity of creatine kinase (CK; a hormone receptor response marker). Similar results were demonstrated in cultured human male or female-derived bone cells (Obs); female Obs responded only to estradiol-17β (E2) whereas male Obs responded only to dihydrotestosterone (DHT) by stimulating DNA, CK, 1α,25 vitamin D hydroxylase (1OHase) mRNA expression and production of 1,25(OH)2D3. Rat epiphyseal cartilage in vivo and its derived cell cultures in vitro responded to both hormones. The sex-specific response of Obs was modified by manipulating the endocrine environment in early development like gonadectomy, neonatally androgenization of female rats and androgen-receptor deficiency in male rats (Tfm). Rat bone marrow (BM) transplanted under the renal capsule formed bone ossicles responding to GS according to the donor gender. Rat demineralized tooth matrix particles (DTM) implanted under the skin induced bone formation responding to GS according to the host gender. Cultured femoral rat bone BM differentiated into Obs responding to GS according to the donor gender, losing sex-specificity after gonadectomy. Human Obs responded sex-specifically to GS via both genomic (DNA and CK) and non-genomic (intracellular Ca++ concentration) binding sites. Male Obs although not responding to estrogens, expressed estrogen receptors suggesting involvement of post- receptor mechanism(s). The less differentiated epiphyseal cartilage cells and the de-differentiated Obs after gonadectomy lost sex-specific responses. In conclusion cultured Obs and bones in vivo respond sex-specifically to GS by different parameters.