Estrogen has protective effects on the skeleton via its inhibition of bone resorption. Mechanisms for these effects and the selectivity to the estrogen receptor alpha (ERalpha) or ERbeta are unclear. The purpose of our study was to determine the impact of the ERalpha on skeletal metabolism using murine models with targeted disruption of the ERalpha and beta. Mice generated by homologous recombination and Cre/loxP technology yielding a deletion of the ERalpha exon 3 were evaluated and also crossed with mice with a disruption of the exon 3 of the ERbeta to result in double ERalpha and ERbeta knockout mice. Skeletal analysis of long bone length and width, radiographs, dual X-ray absorptiometry, bone histomorphometry, micro computerized tomography, biomechanical analysis, serum biochemistry, and osteoblast differentiation were evaluated. Male ERalpha knockout mice had the most dramatic phenotype consisting of reduced bone mineral density (BMD), and bone mineral content (BMC) of femurs at 10 and 16 weeks and 8-9 months of age. Female ERalpha knockout mice also had reduced density of long bones but to a lesser degree than male mice. The reduction of trabecular and cortical bone in male ERalpha knockout mice was statistically significant. Male double ERalpha and ERbeta knockouts had similar reductions in bone density versus the single ERalpha knockout mice suggesting that the ERalpha is more protective than the ERbeta in bone. In vitro analysis revealed no differences in osteoblast differentiation or mineralized nodule formation among cells from ERalpha genotypes. These data suggest that estrogens are important in skeletal metabolism in males; the ERalpha plays an important role in estrogen protective effects; osteoblast differentiation is not altered with loss of the ERalpha; and compensatory mechanisms are present in the absence of the ERalpha and/or another receptor for estrogen exists that mediates further effects of estrogen on the skeleton.