OBJECTIVE Although the beneficial effects of estrogen on bone are well known, the roles of estrogen receptors (ERs) in mediating these effects are not fully understood. METHODS To study the effects of long-term ER alpha deficiency, bone phenotype was studied in aged ER alpha knockout (ERKO) mice. In addition, ERKO osteoclasts and osteoblasts were cultured in vitro. RESULTS Histomorphometric analysis showed that the trabecular bone volume and thickness were significantly increased and the rate of bone formation enhanced in both male and female ERKO mice in comparison to the wild-type animals. In ERKO males, however, the bones were thinner and their maximal bending strengths decreased. Consistent with previous reports, the bones of knockout mice, especially of female mice, were shorter than those of wild-type mice. In addition, the growth plates were totally absent in the tibiae of aged ERKO females, whereas the growth plate cartilages were detectable in wild-type females as well as in all the males. Analysis of cultured bone marrow cells from 10- to 12-week-old mice demonstrated that 17 beta-estradiol could stimulate osteoblastic differentiation of bone marrow cells derived from ERKO mice relatively to the same extent as those derived from wild-type mice. This was demonstrated by increases in synthesis of type I collagen, activity of alkaline phosphatase and accumulation of calcium in cultures. Total protein content was, however, reduced in ERKO osteoblast cultures. CONCLUSIONS These results show altered bone phenotype in ERKO mice and demonstrate the stimulatory effect of estrogen on osteoblasts even in the absence of full-length ER alpha.