Heparan sulfate interacts with a variety of proteins and thus mediates numerous complex biological processes. These interactions critically depend on the patterns of O-sulfate groups within the HS chains that determine binding sites for proteins. In particular the distribution of 6-O-sulfated glucosamine residues influences binding and activity of heparan sulfate dependent signaling molecules. The protein binding domains of heparan sulfate show large structural variability, potentially due to differential expression patterns of heparan sulfate biosynthetic enzymes along with differences in substrate specificity. To investigate if different isoforms of heparan sulfate glucosaminyl 6-O-sulfotransferase (6-OST) give rise to differently sulfated domains, we have introduced mouse 6-OST1, 6-OST2 and 6-OST3 in human embryonic kidney 293 cells and compared the effects of overexpression on heparan sulfate structure. High expression of any one of the 6-OST enzymes resulted in appreciably increased 6-O-sulfation of N-sulfated as well as N-acetylated glucosamine units. The increased 6-O-sulfation was accompanied by a decrease in non-sulfated as well as in iduronic acid 2-O-sulfated disaccharide structures. Furthermore, overexpression lead to an altered heparan sulfate domain structure, the most striking effect was the formation of extended 6-O-sulfated predominantly N-acetylated heparan sulfate domains. Although the effect was most noticeable in 6-OST3 expressing cells, these results were largely independent of the particular 6-OST isoform expressed and mainly influenced by level ofoverexpression.