Gel protein of barley can form a gel layer on the top of the mash cake and thereby can affect the filtration time. The gel protein is formed from D and B hordeins (Moonen et al 1984) that are not hydrolyzed during mashing. During germination cysteine proteinases most probably are in charge of bulk hydrolysis of hordeins. During mashing it has been shown that proteinases belonging to cysteine, aspartic and metalloproteinases affect the amount of soluble protein of the wort. These malt endoproteinases were inactivated when the mash temperature reached 70 C (Jones 1999). The aim was to study malt proteinases that can hydrolyze barley gel protein. Gel protein was extracted from resting barley seeds and the proteinases were extracted from malt. These were then incubated together under different conditions. The hydrolysis reactions were followed by fractionation of the gel protein using SDS-PAGE. The gel protein consisted of D and B hordeins as expected. When analyzed under nonreducing conditions it did not penetrate the gel, indicating a polymeric nature. No degradation or breakage of polymer was detected when the substrate was incubated in the absence of the enzymes. The malt proteinases hydrolyzed the gel protein over a broad range of pH (pH 3.8-6.2). The best pH value for the gel protein hydrolysis was pH 5.0, which is also the pH of germinating barley endosperm. The hydrolysis was partially inhibited by E-64, a cysteine proteinase inhibitor. Class specific inhibitors of the other proteinase classes had no effect on the extent of the hydrolysis. The proteinases started to inactivate after pre incubation at 80 C and were totally inactivated after 15 minutes at 90 C. Hydrogen peroxide, an oxidizing agent also partially prevented the hydrolysis. The results of this study indicate that the gel protein is most easily hydrolyzed by malt endoproteinases at pH 5 and that these may be cysteine proteinases. These enzymes are inactivated at rather high temperatures and somewhat inactivated by oxidizing conditions, which occur during mashing.