Two protein proteinase inhibitors were isolated and purified from the leech Hirudo medicinalis by means of gel filtration and ion-exchange chromatography. They inhibit chymotrypsin, subtilisin and the granulocytic neutral proteases elastase and cathepsin G. They proved to be homogeneous in polyacrylamide and dodecylsulfate gel electrophoresis and by end group analysis; only threonine was found as N-terminal amino acid residue using the dansylation technique. These inhibitors, which we call eglins, are stable in neutral and weakly acid (pH 3) solutions and resist non-specific proteolysis. From the amino acid compositions, a molecular weight of 6 600 - 6 800 is calculated for both inhibitory proteins, which is in good agreement with a value of about 6000 estimated by dodecylsulfate electrophoresis. The eglins contain an unusually large amount of hydrophobic amino acid residues but no methionine, isoleucine or--a rarity--cysteine residues or disulfide bridges. To our knowledge, the eglins are the first examples of proteinase inhibitors of the protein type which are not stabilized by disulfide bridges.