Glycosaminoglycan (GAG) assembly initiates through the formation of a linkage tetrasaccharide region serving as a primer for both chondroitin sulfate (CS) and heparan sulfate (HS) chain polymerization. A possible role for sulfation of the linkage structure and of the constitutive disaccharide unit of CS chains in the regulation of CS-GAG chain synthesis has been suggested. To investigate this, we determined whether sulfate substitution of galactose (Gal) residues of the linkage region or of N-acetylgalactosamine (GalNAc) of the disaccharide unit influences activity and specificity of chondroitin sulfate N-acetylgalactosaminyltransferase-1 (CSGalNAcT-1), a key glycosyltransferase of CS biosynthesis. We synthesized a series of sulfated and unsulfated analogs of the linkage oligosaccharide and of the constitutive unit of CS and tested these molecules as potential acceptor substrates for the recombinant human CSGalNAcT-1. We show here that sulfation at C4 or C6 of the Gal residues markedly influences CSGalNAcT-1 initiation activity and catalytic efficiency. Kinetic analysis indicates that CSGalNAcT-1 exhibited 3.6-, 1.6-, and 2.2-fold higher enzymatic efficiency due to lower K-m values toward monosulfated trisaccharides substituted at C4 or C6 position of Gal1, and at C6 of Gal2, respectively, compared with the unsulfated oligosaccharide. This highlights the critical influence of Gal substitution on both CSGalNAcT-1 activity and specifity. No GalNAcT activity was detected toward sulfated and unsulfated analogs of the CS constitutive disaccharide (GlcA-beta 1,3-GalNAc), indicating that CSGalNAcT-1 was involved in initiation but not in elongation of CS chains. Our results strongly suggest that sulfation of the linkage region acts as a regulatory signal in CS chain initiation.