A five-step isolation procedure has been developed for the purificationof isoforms of hirudin (isohirudins) from whole leeches.The final purification of two thrombin-inhibiting preparations by reversed-phase high-performance liquid chromatography yielded several isoleucine but with either N-terminal valine or isoleucine but with identical inhibitioncharacteristics, i.e. specific thrombin inhibiting activities of 680-720IU/mg and dissociation constant Ki of the thrombin-inhibitor complexes to3 x 10-11 mol/l.The inhibitor with N-terminal isoleucine was designated hirudin PA. Thisinhibitor contains 66 amino-acid residues and has a molecular mass of 7 087 Da. The complete amino-acid sequence of hirudin PA was establishedby automated solid-phase Edman degradation of the native and oxidized inhibitor and two of its tryptic fragments.On the basis of the primary structures two types of thrombin inhibitorsfrom the leech can be distinguished,designated hirudin and hirudin PA.The degree of structural homology of both isoinhibitors is approximately82%; both have a tyrosine-O-sulfate residue near the C-terminus.