AbstractA proteinase inhibitor specific for trypsin was purified from Italian millet (Setaria italica) 170‐fold by extraction with 0.02M HC1, ammonium sulphate fractionation, chromatography on CM‐cellulose and trypsin‐affigel chromatography. The inhibitor with an Mr of 14000 was found to be homogeneous by gel chromatography on Sephadex G‐100 and sodium dodecyl sulphate‐polyacrylamide gel electrophoresis. It reacted with bovine trypsin in a 1:1 stoichiometric fashion. Inhibition of trypsin was nearly double when assayed with benzoyl DL‐arginine p‐nitroanilide as substrate compared to the activity with casein as substrate. The inhibitor affected the tryptic activity in human and bovine pancreatic preparations to an equal extent. Chemical modification studies showed that amino groups, disulphide bridges and sulphydryl groups are essential for the action of the inhibitor. Pretreatment with porcine pepsin or bovine α‐chymotrypsin increased the antitryptic activity of the inhibitor when assayed with the low molecular weight substrate but not with casein.