Nematophagous fungi Pochonia chlamydosporia and P. rubescens colonize endophytically barley roots. During nematode infection, serine proteases are secreted. We have investigated whether such proteases are also produced during root colonization. Polyclonal antibodies against serine protease P32 of P. rubescens cross-reacted with a related protease (VCP1) of P. chlamydosporia, but not with barley proteases. These antibodies also detected an unknown ca. 65-kDa protein, labeled hyphae and appressoria of P. chlamydosporia and strongly reduced proteolytic activity of extracts from fungus-colonized roots. Mass spectrometry (MS) of 32-kDa protein bands detected peptides homologous to VCP1 only in Pochonia-colonized roots. Peptides homologous to barley serine carboxypeptidases were found in 65kDa bands of all roots. RT-PCR detected expression of VCP1 and a new P. chlamydosporia serine carboxypeptidase (SCP1) genes only in fungus-colonized roots. SCP1 shared limited sequence homology with VCP1 and P32. Expression in roots of proteases from nematophagous fungi could be greatly relevant for nematode biocontrol.