Immunohistochemical studies have localized thrombospondin (TSP), a platelet adhesive protein, to the atherosclerotic plaque. To investigate how TSP may become incorporated in the plaque, we evaluated the interaction of TSP with human plasma lipoproteins and apolipoproteins. Chylomicrons, VLDL, LDL, HDL, and apolipoproteins AI, AII, C were immobilized on microtiter plates. Binding to TSP was measured directly with [125I]TSP. Labeled TSP bound saturably to all the plasma lipoproteins tested, showing the highest capacity for binding to VLDL. This binding to VLDL was maximal in the presence of 1 mM CaCl2 and MgCl2 and only partially inhibited with EDTA. The binding was inhibited totally by incubation with fluid-phase lipoproteins, apolipoproteins or anti-TSP monoclonal antibodies. The dissociation constants (Kd) for VLDL and apo C were 153 nM and 150 nM, respectively. Thus, TSP exhibits specific and saturable binding with high affinity to VLDL, perhaps mediated by its surface apo C. This binding may facilitate TSP incorporation into nascent atherosclerotic plaques and delivery of VLDL cholesterol into these lesions.