We purified a trypsin inhibitor, designated therin, from the rhynchobdellid leech Theromyzon tessulatum. Therin was purified to apparent homogeneity by gel-permeation and anion-exchange chromatography followed by reverse-phase HPLC. By a combination of reduction and S-β-pyridylethylation, Edman degradation and electrospray mass spectrometry measurement, the complete sequence of therin (48 amino acid residues ; m/z, 5376.35Ϯ0.22 Da) was determined. Therin exhibits an approximately 30% sequence similarity with peptides of the antistasin-type inhibitors family, i.e. the first and second domains of antistasin, hirustasin, ghilanthen and guamerins (I, II). Therin is a tight-binding inhibitor of trypsin (K i , 45Ϯ 12 pM) and has no action towards elastase or cathepsin G. Furthermore, therin (10 Ϫ6 M) in conjunction with theromin, a Theromyzon thrombin inhibitor (10 Ϫ6 M) significantly diminish the level of human leucocytes activation induced by lipopolysaccharide (10 µg) in a manner similar to that of aprotinin. These data suggest a leech trypsin inhibitor with possible biomedical signifance.