Osteopontin (OPN) is a prominent bone matrix protein that is synthesized by osteoblastic cells. To elucidate the function of OPN in bone we studied the regulated expression of the rat OPN protein during bone formation in vivo and in vitro. OPN mRNA is expressed by preosteoblastic cells early in bone formation, but the highest expression is observed in mature osteoblasts at sites of bone remodelling. A low-phosphorylated, 55-kDa form of OPN is produced by the preosteoblastic cells, whereas osteoblasts produce a highly phosphorylated, 44-kDa protein; the two forms of OPN corresponding to pp69 and pp62 in transformed rat cells. The synthesis of the 55-kDa OPN correlates with the formation of a 'cement' matrix that is synthesized prior to bone deposition, whereas the 44-kDa OPN synthesized by osteoblasts associates rapidly with hydroxyapatite, possibly regulating crystal growth, and may also provide a substratum for osteoclast attachment. Expression of OPN mRNA is upregulated by growth and differentiation factors (PDGF, EGF, TGF-beta and BMP-7/OP-1) and by mechanical stress, which promote bone formation, as well as by osteotropic hormones (retinoic acid and vitamin D3), which can promote bone resorption and remodelling. However, OPN mRNA is down-regulated by bisphosphonates, which abrogate bone resorption. Regulation of OPN expression is, therefore, consistent with a multiplicity of functions for OPN that involve specific structural motifs in both the synthesis and resorption of bone.