To investigate the mechanisms by which megakaryocytes (MKs) may influence bone remodelling, CD34+ cells were cultured for 6, 9 and 12 d with or without 17β-oestradiol (E) and immunolocalized for osteoprotegerin (OPG), receptor activator of nuclear factor (NF)-κB ligand (RANKL) and CD61. Specific protein expression was measured quantitatively by image analysis. Fluorescence-based immunocytochemistry was used to co-localize OPG and RANKL with CD61. OPG and RANKL mRNA was assessed in CD61+ cells with or without E at 24 and 48 h. At 6 d, OPG and RANKL expression was unchanged by E treatment. At 9 d, the E-treated cultures with maturing MKs showed a 1·72-fold (P < 0·01) increase in OPG expression and a 1·8-fold (P < 0·01) reduction in RANKL. Maximal OPG expression was seen at 12 d with a threefold induction of expression (P < 0·001), whilst RANKL levels were further suppressed by 2·3-fold compared with controls (P < 0·001). CD61 co-localized with OPG and RANKL. mRNA data were consistent with that of protein, with a 90-fold induction in OPG expression and a 34-fold suppression of RANKL expression by E (P < 0·001). Thus, E stimulates megakaryocytopoiesis and modulates OPG and RANKL expression, providing evidence that MKs may play a role in bone remodelling and, in particular, in E-induced changes in osteoclastogenesis and bone resorption.