The interaction of human thrombospondin with mucopolysaccharides has been measured quantitatively. Binding of thrombospondin to heparin was examined utilizing an assay employing an 125I-labeled LMW heparin glycosaminoglycan (Mr = 8500). By this means, a class of binding sites was detected that bound approximately 2 moles of LMW heparin per mole of thrombospondin with a Kd of 2.4 nM. The binding stoichiometry of LMW heparin to thrombospondin was confirmed by fluorescence polarization experiments in which thrombospondin bound 3 moles of fluorescamine-labeled LMW-heparin mole protein. A variety of mucopolysaccharides were able to inhibit the interaction of thrombospondin with 125I-LMW heparin, the most effective being heparin sulfate and dermatan sulfate. However, PF4 was found to be an even more potent inhibitor, approximating unfractionated heparin in this respect. The ability of mucopolysaccharides to interact with purified thrombospondin suggests a role for such molecules in the regulation of the biologic activity of thrombospondin, possibly in interactions with connective tissue, such as the subendothelium. Given that there are three binding sites per molecule and that thrombospondin appears to form polymeric aggregates with itself, significant binding energies could be developed.