The thrombin inhibitor hirudin could be obtained from a prepurified preparation by chromatography on DEAE-Sephadex with a specific activity of 10,400 antithrombin-units/mg. The inhibitor proved to be homogeneous by analysis in the ultracentrifuge and TISELIUS-electrophoresis. Hirudin has the following amino acid composition: Asp10 Glu13 Cys6 Ser4 Gly9 Thr4 Ala1 Val3 Leu4 Ile2 Pro3 Phe2 Try2 His1 Lys4. Isoleucine was found to be the N-terminal residue. The polypeptide inhibitor was destroyed by incubation with papain, pepsin and subtilopeptidase A and was resistant to plasmin, chymotrypsin A and trypsin.