We have shown previously that a highly sulfated sequence, GalNAc(4,6-SO4)-GlcA(2SO4)-GalNAc(6SO4), is present at the nonreducing terminal of chondroitin sulfate (CS), and this structure was synthesized from a unique sequence, GalNAc(4SO4)-GlcA(2SO4)-GalNAc(6SO4), by the sulfation with N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase. Uronosyl 2-O-sulfotrasferase (2OST), which transfers sulfate from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to position 2 of GlcA residue of CS, is expected to be involved in the synthesis of these structures; however, the specificity of 2OST concerning recognition of sulfation pattern of the acceptor has largely remained unclear. In the present study, we examined the specificity of 2OST in terms of recognition of sulfation pattern around the targeting GlcA residue. The recombinant 2OST could sulfate CS-A, CS-C and desulfated dermatan sulfate. When 35S-glycosaminoglycans formed from CS-A after the reaction with the recombinant 2OST and [35S]PAPS were subjected to limited digestion with chondroitinase ACII, a radioactive tetrasaccharide (Tetra A) was obtained as a sole intermediate product. The sequence of Tetra A was found to be HexA-GalNAc(4SO4)-GlcA(2SO4)-GalNAc(6SO4) by the enzymatic and chemical reactions. These observations indicate that 2OST transfers sulfate preferentially to the GlcA residue located in a unique sequence, -GalNAc(4SO4)-GlcA-GalNAc(6SO4)-. When oligosaccharides with different sulfation pattern were used as the acceptor, GalNAc(4SO4)-GlcA-GalNAc(6SO4) and GlcA-GalNAc(4SO4)-GlcA-GalNAc(6SO4) were the best acceptors for 2OST among trisaccharides and tetrasaccharides, respectively. These results suggest that 2OST may be involved in the synthesis of the highly sulfated structure found in CS-A.