We have previously shown that cytokine-induced production of interleukin-6 (IL-6) by cultured bone marrow-derived stromal and osteoblastic cells is inhibited by 17 beta-estradiol, and that estrogen withdrawal (ovariectomy) in mice causes an up-regulation of osteoclast development which can be prevented by a neutralizing antibody against IL-6 or estrogen replacement. To directly establish the link between estrogen loss and altered IL-6 production, implied by our earlier studies, we have now compared IL-6 production in ex vivo cultures of bone marrow cells from mice that were sham operated, ovariectomized, or ovariectomized and treated with 17 beta-estradiol. In addition, we have examined the effect of the in vitro withdrawal of estrogens from primary cell cultures of neonatal murine calvaria on IL-6 production. IL-6 production in ex vivo cultures of bone marrow cells maintained in the presence of 1,25-dihydroxyvitamin D3 or PTH was greater in marrow cells from ovariectomized mice than in those from sham-operated animals or ovariectomized animals receiving estrogen replacement. In line with this finding, addition of 17 beta-estradiol to calvaria cell cultures followed by withdrawal of the steroid caused an increase in the amount of IL-6 produced in response to the subsequent stimulation of these cultures with IL-1 or PTH compared to that in cultures that had never been treated with estradiol; when the inactive isomer 17 alpha-estradiol was used, no change in IL-6 production was observed. These results establish that estrogen loss causes an up-regulation of IL-6 production by bone marrow cells and that a similar phenomenon can be elicited in vitro by withdrawal of 17 beta-estradiol from primary cultures of bone cells.