Barley (Hordeum vulgare L.) that is infested with Fusarium head blight (FHB, ‘scab’) is unsuitable for malting and brewing because it may contain mycotoxins and has unacceptable malting quality. Fungal proteinases are apparently often involved in plant-microbe interactions, where they degrade storage proteins, but very little is known about the enzymes that the fungi produce in the infected grain. We have shown previously that one plant pathogenic fungus, Fusarium culmorum, produced subtilisin- and trypsin-like enzymes when grown in a cereal protein medium. To establish whether these proteinases were also synthesized in FHB-infested barley in vivo, field-grown barley was infested as the heads emerged. Extracts were prepared from the grain as it developed and matured and their proteolytic activities were measured with N-succinyl-Ala-Ala-Pro-Phe p -nitroanilide and N-benzoyl-Val-Gly-Arg p -nitroanilide. The heavily infested barleys contained both subtilisin- and trypsin-like activities. These enzymes reacted with antibodies prepared against each of the two F. culmorum proteinases, indicating that those produced in the laboratory cultures and in the field-infested barley were the same. The presence of these proteinases correlated with the degradation of specific buffer-soluble proteins in the infested grains. These enzymes readily hydrolyzed barley grain storage proteins (C- and D-hordeins) in vitro. The presence of these Fusarium proteinases in the barley indicates that they probably play an important role in the infestation, but exactly how and when they function is not clear.