A novel estrogen receptor, estrogen receptor β (ERβ), has recently been cloned from a rat prostate cDNA library. In bone, which is an important target tissue of estrogen, ERα has been reported to be present preferentially in osteoblasts, but the mechanism of action of estrogen in bone is still not known. In the present study, we examined expression of ERβ mRNA in bone. Expression of ERβ mRNA was evident in primary osteoblastic cells isolated from 1-day-old rat calvaria and rat osteosarcoma cells (ROS 17/2.8), and its level was higher than that of ERα mRNA. When osteoblastic cells were cultured for 28 days to induce differentiation into mature osteoblasts capable of forming bone nodules, ERβ mRNA was constantly and highly expressed during the entire culture period. In contrast, the level of ERα mRNA was very low at the beginning of culture and it gradually increased during the differentiation of osteoblastic cells. Various tissues including bone were isolated from 8-week-old rats of both sexes, and total RNA was extracted to compare the tissue distribution of expression levels of ERβ mRNA. In cancellous bone of the distal femoral metaphysis and lumbar vertebra, expression of ERβ mRNA was obvious, and its level was equivalent to those in the uterus and testis, but lower than those in the ovary and prostate. The level of ERβ mRNA in femoral cortical bone was lower than that in cancellous bone. There was no appreciable differences between female and male rats in the distribution and expression levels of ERβ mRNA in bone. These results indicate that ERβ mRNA is highly expressed in osteoblasts in rat bone, suggesting that there is a distinct mechanism of estrogen action mediated by ERβ in bone.