Thrombospondin-1 (TSP1) is a secreted trimeric glycoprotein of 450 kDa with demonstrated effects on cell growth, adhesion and migration. Its complex biological activity is attributed to its ability to bind to cell-surface receptors, growth factors and extracellular-matrix proteins. In this study, we used a ¹²⁵I solid-phase binding assay to demonstrate that TSP1 binds specifically to proteins containing polyhistidine stretches. Based on studies with three different six-histidine-containing recombinant proteins, we derived an average dissociation constant of 5 nM. The binding of ¹²⁵I-labelled TSP1 to these proteins was inhibited by peptides containing histidine residues, with the degree of competition being a function of the number of histidines within the peptide. Binding was not inhibited by excess histidine or imidazole, indicating that the imidazole ring is not sufficient for recognition by TSP1. Heparin was a potent inhibitor of binding with a K_i of 50 nM, suggesting that the heparin-binding domain of TSP1 may be involved in this interaction. This was confirmed by the ability of a recombinant heparin-binding domain of TSP1 to directly compete for TSP1 binding to polyhistidine-containing proteins. Affinity chromatography with a polyhistidine-containing peptide immobilized on agarose revealed that TSP1 in platelet releasates is the major polypeptide retained on the six-histidine-peptide column. We conclude that TSP1 contains a high-affinity binding site for polyhistidine and this is likely to be the molecular basis for the observed binding of TSP1 to histidine-rich glycoprotein. The possibility that other polyhistidine-containing proteins also interact with TSP1 warrants further study.