Heparan, the common unsulfated precursor of heparan sulfate (HS) and heparin, is synthesized on the glycosaminoglycan-protein linkage region tetrasaccharide GlcUA-Gal-Gal-Xyl attached to the respective core proteins presumably by HS co-polymerases encoded by EXT1 and EXT2, the genetic defects of which result in hereditary multiple exostoses in humans. Although both EXT1 and EXT2 exhibit GlcNAc transferase and GlcUA transferase activities required for the HS synthesis, no HS chain polymerization has been demonstrated in vitro using recombinant enzymes. Here we report in vitro HS polymerization. Recombinant soluble enzymes expressed by co-transfection of EXT1 and EXT2 synthesized heparan polymers with average molecular weights greater than 1.7 x 105 using UDP-[3H]GlcNAc and UDP-GlcUA as donors on the recombinant glypican-1 core protein and also on the synthetic linkage region analog GlcUA-Gal-O-C2H4NH-benzyloxycarbonyl. Moreover, in our in vitro polymerization system, a part time proteoglycan, alpha-thrombomodulin, that is normally modified with chondroitin sulfate served as a polymerization primer for heparan chain. In contrast, no polymerization was achieved with a mixture of individually expressed EXT1 and EXT2 or with acceptor substrates such as N-acetylheparosan oligosaccharides or the linkage region tetrasaccharide-Ser, which are devoid of a hydrophobic aglycon, suggesting the critical requirement of core protein moieties in addition to the interaction between EXT1 and EXT2 for HS polymerization.