We report the results of an immunogold electron microscopical analysis on microfibrils from the arterial subendothelium showing that thrombospondin (TSP) is present on 40 nm-diameter structures joining 8-10 nm-diameter microfibrils containing fibrillin. They differ from type VI collagen which forms 3-5 nm-diameter microfibrils. TSP containing microfibrils (TSP-MF) extracted from human umbilical arteries did not contain fibrillin or type VI collagen. Blood platelet interactions with TSP-MF were not modified by anti-fibrillin or anti-type VI collagen antibodies. In situ, vWF was bound to cross-linked microfibrils, at the level of their 40 nm junction, and a double-labeling with the anti-thrombospondin and anti-vWF antibodies was observed. In vitro, vWF binding to TSP-MF was not inhibited by anti-fibrillin or anti-type VI collagen antibodies. These results suggest a structural and functional heterogeneity of microfibrils and emphasize the role of TSP-MF in the thrombogenicity of the subendothelium.