Endostatin is an endogenous inhibitor of angiogenesis. Although several endothelial cell surface molecules have been reported to interact with endostatin, its molecular mechanism of action is not fully elucidated. We used surface plasmon resonance assays to characterize interactions between endostatin, integrins, and heparin/heparan sulfate. alpha5beta1 and alphavbeta3 integrins form stable complexes with immobilized endostatin (KD=approximately 1.8x10(-8) M, two-state model). Two arginine residues (Arg27 and Arg139) are crucial for the binding of endostatin to integrins and to heparin/heparan sulfate, suggesting that endostatin would not bind simultaneously to integrins and to heparan sulfate. Experimental data and molecular modeling support endostatin binding to the headpiece of the alphavbeta3 integrin at the interface between the beta-propeller domain of the alphav subunit and the betaA domain of the beta3 subunit. In addition, we report that alpha5beta1 and alphavbeta3 integrins bind to heparin/heparan sulfate. The ectodomain of the alpha5beta1 integrin binds to haparin with high affinity (KD=15.5 nM). The direct binding between integrins and heparin/heparan sulfate might explain why both heparan sulfate and alpha5beta1 integrin are required for the localization of endostatin in endothelial cell lipid rafts.