In order to identify the regions of antithrombin that interact with heparin and thrombin, it was degraded with CNBr and the activities of the isolated products were investigated. These fragments did not exhibit direct thrombin-neutralizing activity; however, one unique fragment was found to bind to heparin-Sepharose and also to interfere with the inhibition of thrombin by intact antithrombin. This fragment was identified as the one consisting of three disulphide-linked polypeptide chains containing residues 1-17, 104-251 and 424-432. At a concentration of 46 nM, this product decreased the heparin-enhanced thrombin-inhibitory activity of antithrombin by half, and completely abolished this inhibition when above 300 nM. In the absence of heparin, the action of antithrombin was not completely nullified by the fragment, even when present at relatively high concentrations. At a given fragment concentration, the extent of inhibition was independent of antithrombin concentration over the range tested. It was found that the fragment decreased the second-order rate constant for the antithrombin-thrombin reaction. Reduction and alkylation of the fragment showed that the above properties reside primarily in the peptide with residues 104-251. It is concluded that this peptide possesses portions of the antithrombin molecule that bind to heparin as well as to a site on thrombin.