Thrombospondin (TS), a protein first described in platelets, was recently shown to be synthesized and secreted by endothelial cells, fibroblasts, and smooth muscle cells. The presence of TS in the extracellular matrix of cultured cells has prompted us to examine the associations of this protein with matrix macromolecules. Interactions of TS with both matrix and serum proteins were tested using an enzyme-linked immunosorbent assay. With this assay we assessed the binding of TS in solution to proteins adsorbed to polystyrene microtiter plates. Among collagens, platelet TS bound to type V but not to types I, III, or IV. This selective interaction was confirmed in experiments using proteins linked to cyanogen bromide-activated Sepharose. TS released from platelets in response to thrombin activation, as well as that secreted by endothelial cells in culture, bound to type V but not to type I collagen-Sepharose. No binding was observed to denatured type V collagen-Sepharose. The binding region for type V collagen was located in a chymotrypsin-produced fragment of TS with chains of Mr = 70,000, after reduction. Interactions of TS with a number of other proteins, including fibronectin, fibrinogen, and laminin, could be demonstrated using the enzyme-linked immunosorbent assay technique but the interpretation of these findings is difficult since comparable binding to protein-Sepharose was not always observed. Our findings suggest that both the extravascular distribution and function of TS in vivo may involve an interaction with type V collagen.