To study a hypothesis that thrombospondin (TSP) might function as an agglutinin in platelet aggregation, we designed two experiments. First, we prepared fibrinogen-coated agarose beads (fbg-beads) as a model of platelets, and subjected them to aggregometry using TSP as an inducer. TSP induced agglutination of fbg-beads in a dose-dependent manner. Calcium (Ca) and Magnesium (Mg) were necessary for the agglutination, and the aggregability was dependent on the concentration of Ca. These results confirmed the function of TSP as an agglutinin, suggesting some characteristics of the fbg-TSP interaction as well. Secondly, a variety of platelets were subjected to TSP-induced aggregation assay. Both gel-filtrated and washed-platelets were aggregated by TSP in a dose dependent manner and dissociated with EDTA. The same aggregation was observed in formalin-fixed platelets. Both Ca and Mg were required for the aggregation, and the maximum aggregation rate was dependent on the Ca concentration. Ca seemed to regulate the capacity as well as the affinity of the binding sites for TSP on platelets. Fibrinogen and some aminosugars inhibited the aggregation. These data suggest TSP may function as an agglutinin of platelets, and Ca may regulate the interaction between platelets and TSP. As one of the candidates for the receptor for TSP on platelet, fbg-GPIIb/IIIa was suggested because of the similarity between fbg-beads and platelets aggregation induced by TSP, and the Ca-dependency in both the GPIIb/IIIa induction and the TSP-induced platelet aggregation.