A novel estrogen receptor, estrogen receptor beta (ERbeta), has recently been cloned from a rat prostate cDNA library. In bone, which is an important target tissue of estrogen, ER alpha 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 ERbeta mRNA in bone. Expression of ERbeta 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 ER alpha mRNA. When osteoblastic cells were cultured for 28 days to induce differentiation into mature osteoblasts capable of forming bone nodules, ERbeta mRNA was constantly and highly expressed during the entire culture period. In contrast, the level of ER alpha 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 ERbeta mRNA. In cancellous bone of the distal femoral metaphysis and lumbar vertebra, expression of ERbeta 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 ERbeta 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 ERbeta mRNA in bone. These results indicate that ERbeta mRNA is highly expressed in osteoblasts in rat bone, suggesting that there is a distinct mechanism of estrogen action mediated by ERbeta in bone.