Basement membrane laminin (laminin-1) is a multidomain glycoprotein that interacts with itself, heparin and cells. The interaction with heparin/heparan sulfate proteglycans is thought to be important for the architectural formation of basement membranes and adhesion to cells. The major heparin binding site has been known to reside in the long arm globular domain (G domain). The G domain is in turn subdivided into five subdomains (G1-G5). In order to localize the heparin binding regions further, recombinant G domains (rG and rG5) were expressed in Sf9 insect cells using baculovirus expression vector. By the limited proteolysis of recombinant G domains followed by either heparin affinity HPLC or overlay with radiolabeled heparin, the relative affinity of each subdomain to heparin was assigned as G1>G2 = G4>G5>G3, such that G1 bound strongly and G3 not at all. Since the activity in G1-G3 is cryptic in intact laminin long arm [Sung, U., O'Rear, J. J. & Yurchenco, P. D. (1993) J. Cell Biol. 123, 1255-1268], the active heparin binding site of G domain appears to be located in G4 and proximal G5.