Two human GlcNAc-6-sulfotransferases, CHST2 and HEC-GlcNAc6ST, have been recently identified as possible contributors to the inflammatory response by virtue of their participation in L-selectin ligand biosynthesis. Selective inhibitors would facilitate their functional elucidation and might provide leads for antiinflammatory therapy. Here we investigate the critical elements of a disaccharide substrate that are required for recognition by CHST2 and HEC-GlcNAc6ST. A panel of disaccharide analogues, bearing modifications to the pyranose rings and aglycon substituents, were synthesized and screened for substrate activity with each enzyme. Both GlcNAc-6-sulfotransferases required the 2-N-acetamido and 4-hydroxyl groups of a terminal GlcNAc residue for conversion to product. Both enzymes tolerated modifications to the reducing terminal pyranose. Key differences in recognition of an amide group in the aglycon substituent were observed, providing the basis for future glycomimetic inhibitor design.