Conformational changes induced in thrombospondin-1 by removal of calcium regulate interactions with some ligands of its N-modules. Because calcium binds primarily to elements of the C-terminal signature domain of thrombospondin-1, which are distant from the N-modules, such regulation was unexpected. To clarify the mechanism for this regulation, we compared ligand binding to the N-modules of thrombospondin-1 in the full-length protein and recombinant trimeric thrombospondin-1 truncated prior to the signature domain. Three monoclonal antibodies were identified that recognize the N-modules, two of which exhibit calcium-dependent binding to native thrombospondin-1 but not to the truncated trimeric protein. These antibodies or calcium selectively modulate interactions of fibronectin, heparin, sulfatide, alpha3beta1 integrin, tumor necrosis factor-alpha-stimulated gene-6 protein, and, to a lesser extent, alpha4beta1 integrin with native thrombospondin-1 but not with the truncated protein. These results indicate connectivity between calcium binding sites in the C-terminal signature domain and the N-modules of thrombospondin-1 that regulates ligand binding and functional activities of the N-modules.