We investigated the effects of 1,25-dihydroxyvitamin D3 on the synthesis of osteopontin, a phosphorylated cell attachment glycoprotein, in ROS 17/2.8 cells, a clonal osteoblast-like rat osteosarcoma cell line. We observed a dose dependent increase in uptake of [32P04] into osteopontin secreted into the medium. An increased incorporation of [35S] -methionine into secreted osteopontin suggested the effect was that of increased protein biosynthesis. Using a radioimmunoassay we demonstrated a dose dependent increase in the amount of secreted osteopontin, an increase which could be blocked by Actinomycin D, in response to 1,25-dihydroxyvitamin D3. These results suggest that the hormonal form of vitamin D regulates the biosynthesis of osteopontin, possibly at the level of transcription. © 1987, Gustav Fischer Verlag · Stuttgart · New York. All rights reserved.