The binding of pentasaccharide heparin to antithrombin induces a conformational change that is transmitted to the reactive center loop and increases the rate of inhibition of factor Xa by approximately 300-fold. The mechanism of such transmission is not known. To test the role of residues 134-137, which link helix D to beta-sheet A, in this signal transduction, we created variant antithrombins in which we removed amino acids 134-137 stepwise and cumulatively. Although the deletions did not compromise the fundamental ability of antithrombin to bind to heparin or to inhibit target proteinases thrombin and factor Xa, they did largely decouple conformational changes in the heparin-binding site from conformational activation of the reactive center loop. Because the variant with only Ala(134) removed was as compromised as variants with larger deletions, yet the variant with Ser(137) removed was normal, we concluded that the length of the linker is less important than the precise interrelationship between residues in this region and other residues involved in conformational activation of antithrombin.