In this study we have shown by both immunofluorescence and immunoprecipitation techniques that human osteoblasts and osteosarcoma cells synthesize and secrete thrombospondin, a 450-kDa glycoprotein initially found in platelets. Immunofluorescence with a mouse monoclonal antibody to human platelet thrombospondin yielded specific granular staining within the cytoplasm of human osteoblasts. SDS/polyacrylamide gel electrophoresis analysis of immunoprecipitates obtained with polyclonal and monoclonal anti-thrombospondin antibodies allows the identification of thrombospondin in the cellular lysates and the culture media of biosynthetically labelled osteoblasts and osteosarcoma cells. Kinetic and dose/response studies of osteoblasts and of two osteosarcoma cell lines (MG-63, SaOs-2) were performed to assess the ability of these cells to adhere to thrombospondin and type-I collagen. Thrombospondin promoted the attachment of human osteoblasts whereas it inhibited the adhesion of MG-63 and SaOs-2 cells, both when it was directly adsorbed to plastic and when it was bound to type-I collagen. Therefore osteoblasts and osteosarcoma cells may be valuable tools to study the role of thrombospondin in cell adhesion.