A saline extract of hard winter wheat containing a-amylase inhibitors was fractionated by ammonium sulfate precipitation and chromatography on DEAE-Sephacel and BioGel P-100 Two amylase isozymes (Amy-1 and Amy-2) from the rice weevil, Sitophilus oryzae (L.), differed in their sensitivity toward two fractions, P-100-1 and P-100-2, that eluted from the BioGel column. Inhibitor zymograms showed that P-I00-1 and P-I00-2 were heterogeneous and contained at least four and seven proteins, respectively, with inhibitory activity against Amy-2. P-100-1 and P-I00-2 were further fractionated by high performance liquid chromatography on an Aquapore AX-300 column and analyzed by electrophoresis under dissociating and nondissociating conditions. Based on elution profiles from BioGel P-100 and results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, components of P-100-2 are apparently dimers. M, estimates of subunit size varied depending on denaturing conditions (14 kDa [kilodalton] by SDS-PAGE and 12 kDa with an SDS-PAGE system containing 8 M urea). Higher molecular weight inhibitors in P-100-1 dissociated into two components with M, values of 14 and 15 kDa by SDS-PAGE. A mechanism of action of these potential cereal resistance factors is postulated.