Heparin inhibits blood coagulation by binding to the protease inhibitor antithrombin, thus promoting inactivation of the protease "factors" of the coagulation cascade mechanism. The article provides an overview of the studies, by different research groups, that led to the structural elucidation of the antithrombin-binding region of heparin. These studies were triggered by the finding that only a fraction of heparin molecules were capable of binding with high affinity to antithrombin, and further that this fraction essentially accounted for the anticoagulant activity of the unfractionated material. Oligosaccharides obtained by selective, partial depolymerization of heparin were fractionated on immobilized antithrombin, and the smallest high-affinity molecules recovered were subjected to structural analysis, in conjunction with various modification steps. The results were interpreted in terms of a binding site for antithrombin constituted by a pentasaccharide sequence with an internal unique 3-O-sulfated glucosamine unit, in addition to sugar residues and sulfate groups present elsewhere also in the polysaccharide. The structure/function relations deduced were verified by chemical synthesis of several pentasaccharide variants with the predicted bioactivities.