We have analyzed the interactions of platelets and platelet-secreted proteins with proteins bound to glass substrata. Using a newly developed radioactive crosslinking reagent, N-succinimidyl-3-[(2-nitro-4-azidophenyl)-2-aminoethyldithio] propionate, we observed crosslinking between thrombospondin released from the platelets and surface-bound fibronectin or collagen. The crosslinking was selective and specific, since thrombospondin showed little crosslinking to substratum-bound bovine serum albumin or ovalbumin, even though it bound to these surfaces. Furthermore, although albumin and fibrinogen released by platelets bound to fibronectincoated substrata as well as did thrombospondin, these two proteins crosslinked at much lower levels than seen for thrombospondin. Interaction between fibronectin and thrombospondin was confirmed by affinity chromatography. These results suggest that thrombospondin and fibronectin may interact during platelet-substratum adhesion or during platelet-platelet aggregation, or both.