An inhibitor affecting subtilisin [EC 3.4.21.14] was isolated from barley (Hordeum vulgare L cv. Kikaihadaka) by extraction with 1% sodium chloride, fractionation with ammonium sulfate, chromatography on CM- and DEAE-cellulose columns, and gel filtration on Sephadex G-100. The final preparation appeared to be homogeneous on the basis of poly aerylamide gel electrophoresis; the inhibitory activity against subtilisin was increased about 140-fold during purification. This inhibitor was a protein having a molecular weight of about 20, 000, and containing 177 amino acid residues. Both the amino- and carboxyl-terminal residues were alanine. The inhibitor inactivated subtilisin, probably by formation of an enzyme-inhibitor complex in a molar ratio of 1: 1, but had little or no effect on the activities of other enzymes tested. The dissociation constant of the subtilisin-inhibitor complex was 1.5x10−10M. The inhibitor appears to be distinct from the barley microbial proteinase isoinhibitors reported by Mikola and Suolinna, in respect of most of its physicochemical and inhibitory properties.