Estrogen receptor-α (ERα) is crucial for the adaptive response of bone to loading but the role of endogenous estradiol (E2) for this response is unclear. To determine in vivo the ligand dependency and relative roles of different ERα domains for the osteogenic response to mechanical loading, gene targeted mouse models with (i) a complete ERα inactivation (ERα(-/-) ), (ii) specific inactivation of activation function 1 (AF-1) in ERα (ERαAF-1(0) ), or (iii) specific inactivation of ERαAF-2 (ERαAF-2(0) ) were subjected to axial loading of tibia, in the presence or absence (ovariectomy, ovx) of endogenous E2. Loading increased the cortical bone area in the tibia mainly as a result of an increased periosteal bone formation rate (BFR) and this osteogenic response was similar in gonadal intact and ovx mice, demonstrating that E2 (ligand) is not required for this response. Female ERα(-/-) mice displayed a severely reduced osteogenic response to loading with changes in cortical area (-78±15%, p<0.01) and periosteal BFR (-81±9%, p<0.01) being significantly lower than in wild type (WT) mice. ERαAF-1(0) mice also displayed a reduced response to mechanical loading compared with WT mice (cortical area -40±11%, p<0.05 and periosteal BFR -41±8%, p<0.01), while the periosteal osteogenic response to loading was unaffected in ERαAF-2(0 ) mice. Mechanical loading of transgenic estrogen response element (ERE)-luciferase reporter mice did not increase luciferase expression in cortical bone, suggesting that the loading response does not involve classical genomic ERE-mediated pathways. In conclusion, ERα is required for the osteogenic response to mechanical loading in a ligand-independent manner involving AF-1 but not AF-2. © 2012 American Society for Bone and Mineral Research.