Recently, it has been shown that a deficiency in ChGn-1 (chondroitin N-acetylgalactosaminyltransferase-1) reduced the numbers of CS (chondroitin sulfate) chains, leading to skeletal dysplasias in mice. Although these results indicate that ChGn-1 regulates the number of CS chains, the mechanism mediating this regulation is not clear. ChGn-1 is thought to initiate CS biosynthesis by transferring the first GalNAc (N-acetylgalactosamine) to the tetrasaccharide in the protein linkage region of CS. However, in vitro chondroitin polymerization does not occur on the non-reducing terminal GalNAc-linkage pentasaccharide structure. In the present study we show that several different heteromeric enzyme complexes composed of different combinations of four chondroitin synthase family members synthesized more CS chains when a GalNAc-linkage pentasaccharide structure with a non-reducing terminal 4-O-sulfation was the CS acceptor. In addition, C4ST-2 (chondroitin 4-O-sulfotransferase-2) efficiently transferred sulfate from 3'-phosphoadenosine 5'-phosphosulfate to position 4 of non-reducing terminal GalNAc-linkage residues, and the number of CS chains was regulated by the expression levels of C4ST-2 and of ChGn-1. Taken together, the results of the present study indicate that C4ST-2 plays a key role in regulating levels of CS synthesized via ChGn-1.