Thrombospondin (TSP) is a 450‐kDa glycoprotein synthesized and secreted by human MG‐63 osteoblastic cells. In this study, we have first studied the effect of α‐thrombin on TSP expression by human MG‐63 cells. In situ hybridization indicated that TSP mRNA level in thrombin‐treated MG‐63 cells was increased when compared to unstimulated cells. As judged by immunofluorescence, thrombin‐treatment of MG‐63 cells resulted in increased cell surface expression of TSP when compared to quiescent cells. Because thrombin stimulates proliferation of osteoblastic cells, the involvement of TSP in proliferation of thrombin‐stimulated osteoblastic cells was then investigated using a serum‐free mitogenesis assay. Both α‐thrombin (0.01 to 0.15 U/ml) and TSP (5 to 600 ng/ml) caused a dose‐dependent increase in [³H]thymidine incorporation by MG‐63 cells. Proliferation of osteoblastic cells induced by α‐thrombin or TSP was specifically and totally inhibited by anti‐TSP monoclonal antibodies (3–10 μg/ml) or by indomethacin (1 μM), an inhibitor of prostaglandin synthesis. Anti‐TSP antibodies which inhibited cell proliferation also inhibit TSP expression to the surface of these cells. Our experiments support the existence of a mechanism whereby TSP bound to the cell surface of thrombin‐treated MG‐63 cells stimulates secretion of prostaglandins which, in turn, allow cell proliferation to proceed.