Bone sialoprotein (BSP) is a major protein in the mineralized matrix of bone and dentine. To study the relationship between the expression of BSP and the formation of mineralized connective tissues, a cDNA probe to rat BSP was prepared for in situ hybridization analysis of developing fetal rat bones and teeth. When used for Northern hybridization analysis of rat bone marrow cells induced to differentiate into osteogenic cells by dexamethasone, the BSP cDNA revealed a specific induction of 1.6- and 2.0-kb mRNA species of BSP. In tissue sections a strong hybridization signal associated with osteoblasts was observed in areas of endochondral bone formation in the long bone metaphysis and condylar cartilage, and in the intramembranous bone of the calvaria and mandible. Hybridization reflecting a lower degree of expression was evident in cells of the transitional zone of mineralizing cartilage and in odontoblasts forming incisor dentine. Expression of BSP was also demonstrated in the hypertrophic cartilage cells in the long bone and condylar process. In contrast, expression of BSP could not be detected in the reserve or proliferative chondrocytes, fibroblasts and muscle cells. These studies demonstrate that the expression of BSP in bones and teeth is essentially restricted to cells directly involved in the formation of mineralizing connective tissue matrices, indicating that BSP has a specific role in biological mineralization and that it is a useful marker of bone formation.