The fungal disease Fusarium head blight occurs on wheat (Triticum spp.) and barley (Hordeum vulgare L.) and is one of the worldwide problems of agriculture. It can be caused by various Fusarium species. We are characterizing the proteinases of F. culmorum to investigate how they may help the fungus to attack the grain. A trypsin-like proteinase has been purified from a gluten-containing culture medium of F. culmorum. The enzyme was maximally active at about pH 9 and 45 degrees C, but was not stable under those conditions. It was stabilized by calcium ions and by the presence of other proteins. The proteinase was most stable at pH 6-7 at ambient temperatures, but was quickly inactivated at 50 degrees C. It was strongly inhibited by p-amidino phenylmethylsulfonyl fluoride (p-APMSF), and soybean trypsin and Bowman-Birk inhibitors, and it preferentially hydrolyzed the peptide bonds of the protein substrate beta-purothionin on the C-terminal side of Arg (mainly) and Lys residues. These characteristics show that it is a trypsin-like proteinase. In addition, its N-terminal amino acid sequence was 88% identical to that of the F. oxysporum trypsin-like enzyme. The proteinase hydrolyzed the D hordein and some of the C hordeins (the barley storage proteins). This enzyme, and a subtilisin-like proteinase that we recently purified from the same organism, possibly play roles in helping the fungus to colonize grains.