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ABSTRACT: This study investigated whether 17beta-estradiol (E2) may have different effects on osteoblasts derived from estrogen-deficient ovariectomized (OVX) mice compared to sham-operated normal animals. We studied the specific effects of 17beta-estradiol on the differentiation and function of cultured osteoblasts derived from these groups of animals, with or without estrogen replacement treatment. One-month-old mice were ovariectomized or sham-operated, and treated (every second day) for 4 weeks with 0.5 mg/kg 17beta-estradiol or with vehicle alone. At the end of the experiment, bones were removed for primary osteoblast cultures or for morphological and chemical evaluation. In cells from untreated OVX animals, alkaline phosphatase (ALP) specific activity was reduced, while collagen production and mineralization were unchanged when compared to cells from controls. In vivo estrogen pretreatment of the OVX mice elevated ALP activity and mineralization of the cells, while collagen production was reduced. The addition of 17beta-estradiol to the culture medium increased ALP activity, collagen production, and mineralization by all cultured osteoblasts, except in those derived from sham-operated, estrogen-pretreated mice, where these features remained unchanged. Osteocalcin production was unchanged. Addition of testosterone or 1,25(OH)2D3 to the culture medium induced changes that differed among the groups depending on the source of the cultured cells. It seems that ovariectomy in mice prior to culture affected the phenotype of the cultured osteoblasts and their response to estradiol, testosterone, and 1,25(OH)2D3, depending on whether animals were pretreated with estradiol or not. These results imply that the animal's estrogen status prior to culture can influence the response to estrogens; this finding may have important implications for hormone replacement therapy (HRT) in postmenopausal women.