Glycosaminoglycan heparan sulphate interacts with a variety of proteins, such as growth factors, cytokines, enzymes and inhibitors and, thus, influences cellular functions, including adhesion, motility, differentiation and morphogenesis. The interactions generally involve saccharide domains in heparan sulphate chains, with precisely located O-sulphate groups. The 6-O-sulphate groups on glucosamine units, supposed to be involved in various interactions of functional importance, occur in different structural contexts. Three isoforms of the glucosaminyl 6-O-sulphotransferase (6-OST) have been cloned and characterized [H. Habuchi, M. Tanaka, O. Habuchi, K. Yoshida, H. Suzuki, K. Ban and K. Kimata (2000) J. Biol. Chem. 275, 2859-2868]. We have studied the substrate specificities of the recombinant enzymes using various O-desulphated poly- and oligo-saccharides as substrates, and using adenosine 3'-phosphate 5'-phospho[(35)S]sulphate as sulphate donor. All three enzymes catalyse 6-O-sulphation of both -GlcA-GlcNS- and -IdoA-GlcNS- (where GlcA represents D-glucuronic acid, NS the N-sulphate group and IdoA the L-iduronic acid) sequences, with preference for IdoA-containing targets, with or without 2-O-sulphate substituents. 6-OST1 showed relatively higher activity towards target sequences lacking 2-O-sulphate, e.g. the -GlcA-GlcNS- disaccharide unit. Sulphation of such non-O-sulphated acceptor sequences was generally favoured at low acceptor polysaccharide concentrations. Experiments using partially O-desulphated antithrombin-binding oligosaccharide as the acceptor revealed 6-O-sulphation of N-acetylated as well as 3-O-sulphated glucosamine residues with each of the three 6-OSTs. We conclude that the three 6-OSTs have qualitatively similar substrate specificities, with minor differences in target preference.