We have synthesized the pentasaccharide representing the binding site of heparin to AT III. This pentasaccharide contains a 3-0-sulfate group on one of its glucosamine units. A pentasaccharide analogue lacking this 3-0-sulfate group and two different tetrasaccharides, each being part of the pentasaccharide sequence with AT III affinity, have also been synthesized. The pentasaccharide with high affinity for AT III also shows high antifactor Xa activity and, in vivo, antithrombotic activity. Removal of glucosamine at the nonreducing end maintains partial binding capacity as well as partial antifactor Xa activity.