Thrombospondin is a 450‐kDa glycoprotein secreted by a variety of cells including endothelial cells, fibroblasts and platelets. The aim of this study was to compare the structural and immunological properties of human endothelial, fibroblast and platelet thrombospondins. All three thrombospondins were purified, digested with thermolysin, and the subsequent thermolysin‐generated fragments isolated on a Superose 12 gel‐permeation column using non‐denaturating conditions. Each isolated proteolytic fragment of thrombospondins was then detected using either a radioimmunoassay with a polyclonal antibody or an enzyme‐linked immunosorbent assay with three monoclonal antibodies (P10, MA‐I, MA‐II) directed against different epitopes of whole platelet thrombospondin. The fragmentation pattern of human endothelial thrombospondin consists of six major thermolysin‐generated fragments (135–110, 98–82, 54–47, 25–20, 18–15 and 10 kDa) having molecular masses very similar to those observed with human fibroblast thrombospondin (115–100, 92–80, 54–49, 27–21, 17–13 and 12–10 kDa). Treatment of platelet thrombospondin with thermolysin only generated four proteolytic fragments having molecular masses of 110, 50, 25 and 12/10 kDa respectively. All these proteolytic fragments of endothelial, fibroblast and platelet thrombospondins were recognized by a polyclonal antibody. Monoclonal antibodies MA‐I and P10 essentially recognized two proteolytic fragments (135–110, 98–82 kDa) of endothelial and fibroblast (115–100, 92–80 kDa) thrombospondins, and the 110‐kDa fragment of platelet thrombospondin. Monoclonal antibody MA‐II recognized three proteolytic fragments (54–47, 25–20, 18–15 kDa) of endothelial and fibroblast (54–49, 27–21, 17–13 kDa) thrombospondins, and two fragments (50, 25 kDa) of platelet thrombospondin, different from those detected by P10 an MA‐I. The results clearly demonstrate that, under non‐denaturating conditions, endothelial and fibroblast thrombospondins are structurally different from platelet thrombospondin since two fragments of endothelial thrombospondin (98–82, 18–15 kDa), equivalent to those of fibroblast thrombospondin (92–80, 17–13 kDa), are not released from platelet thrombospondin after thermolysin treatment. These three forms of thrombospondin are, however, immunologically indistinguishable. To investigate further the structural differences observed between platelet and the two other forms of thrombospondin, their degree of polymerization was compared. Prior to thermolysin treatment, the three forms of thrombospondin were separated into several oligomers ranging from 450 kDa to 3300 kDa when injected onto a Superose 6 gel‐permeation column. In the presence of thermolysin, platelet thrombospondin oligomers were all digested whereas the 3300 kDa oligomer of endothelial or fibroblast thrombospondin was unaffected. All these oligomers were recognized by a polyclonal antibody and three monoclonal antibodies directed against platelet thrombospondin. Such a resistance to proteolysis of endothelial and fibroblast thrombospondins does explain the structural differences observed in this study, and therefore suggests that the conformational properties of these two forms of thrombospondin are different from those of platelet thrombospondin.