In order to form worts that are acceptable for brewing, a partial 'solubilization' of the barley storage proteins must occur during malting and mashing. This solubilization is carried out by aspartic proteinases that occur in the ungerminated barley and by some of the many cysteine and metalloproteinases that form in the grain during malting. The activities of certain of these proteinases, especially those of the cysteine class enzymes, are regulated by the presence of endogenous barley and malt proteinase inhibitors. We are studying these endoproteinases and their inhibitors to define how the protein hydrolysis system operates so that it can be more precisely regulated to produce improved worts. In this study, malt enzymes (E) and inhibitors (I) were extracted from barley and malt and treated in various ways. The treated samples were then passed through a P-30 chromatography column, which separates biopolymers on the basis of their molecular sizes. This allowed us to ascertain whether the inhibitors were complexed with the proteinases (large, voided the column) or were free (small, retarded on the column). The experiments showed that as soon as the malts were dissolved, enzyme-inhibitor complexes (EI) were present. Some or all of these complexes may already have existed within the malt kernels, or they might have formed when the dissolved E and I molecules interacted. The EI complexes dissociated when heated to boiling, but not when subjected to several other rigorous treatments, indicating that the inhibitors were bound tightly to the proteinases. When free I was prepared by heat-degrading the EI complex and it was added to a solution containing free E molecules, the E and I readily interacted, showing that the I molecules retained their abilities to interact, even after being boiled. In contrast to malt extracts, all of whose I molecules were complexed with E, some free I was present in barley extracts, probably because the barley did not contain sufficient E to complex with all of the I that was present. By collecting EI by chromatography, dissociating it, and separating the resultant I compounds, an 'affinity' method was devised for concentrating the inhibitors. This method has been used as a first step for purifying relatively large amounts of a previously unstudied proteinase inhibitor. While it has not yet been possible to purify E that is free of I by using relatively benign procedures (our original goal), the knowledge gained by these studies has helped to clarify our view of how the E and I molecules work together to degrade proteins during malting and mashing. This knowledge is essential for developing future barley cultivars that have improved proteinase activities and for understanding how various brewing methodologies affect the protein and amino acid contents of worts.