Kringle 5 (K5), located outside of angiostain (K1-4) in human plasminogen, displays more potent antiangiogenic activity on endothelial cell proliferation than angiostatin itself. Using a yeast two-hybrid system in vivo, we have recently identified Rgl2 (guanine nucleotide dissociation stimulator (RalGDS)-like factor 2) as a binding protein of human K5. In order to confirm in vitro protein interaction between K5 and Rgl2, we developed bacterial recombinant expression systems for them. K5 and Rgl2 proteins were expressed in high yields and purified into pure forms with His tags and GST fusion, respectively. GST-pull down experiments clearly demonstrated that K5 interacts specifically with Rgl2 in vitro. These results indicate that Rgl2 functions as a receptor protein for K5 in vitro as well as in vivo, leading to anti-angiogenesis through regulating Ras signaling pathways.