Thrombospondin (TBSP) is a 450 kDa glycoprotein secreted by a wide range of cells including platelets, endothelial cells and fibroblasts. Using non-denaturating conditions, we recently reported that platelet TBSP was structurally different from endothelial and fibroblast TBSP (P. Clezardin et al., Eur. J. Biochem., 1986, 159, 569-579). The aim of this study was to compare the structure of TBSP purified from platelets, endothelial cells and fibroblasts using denaturating conditions. Moreover, the interaction of fibrinogen with these three forms of TBSP was also investigated. TBSPs were first purified by heparin-Sepharose and immunoaffinity chromatography followed by Mono O anion-exchange chromatography on a FPLC system. Thermolysin digests of purified TBSPs were analysed by SDS-polyacrylamide gel electrophoresis under reducing conditions and the subsequent electrophoresed proteolytic fragments identified by Coomassie and silver staining. The interaction of undigested and digested TBSPs with solid-phase adsorbed fibrinogen was investigated by enzyme-linked immunosorbent assay using an anti-TBSP monoclonal antibody (P10). when using Coomassie staining, a 70 kDa proteolytic fragment of thermolysin-treated platelet TBSP was absent from the endothelial and fibroblast TBSP digests. Moreover, a 18 kDa fragment from thermolysin-treated endothelial and fibroblast TBSP was undetectable in the platelet TBSP digest when using silver staining on SDS-polyacrylamide gels. The binding of undigested TBSPs to solid-phase adsorbed fibrinogen was different from that obtained with digested TBSPs. These results indicate that the observed structural differences might induce functional differences between platelet and the two other forms of TBSP.