Pyrenophora graminea is the causal agent of barley leaf stripe. A mutant of the fungus (T2) was obtained by insertional mutagenesis from the highly virulent isolate I2 by random insertion of a plasmid that has no homology with the fungal genome. A marked reduction of disease incidence was observed in two barley cultivars susceptible to leaf stripe, Nudinka and Mirco, when inoculated with the isogenic insertional mutant T2 in comparison with P. graminea isolate I2. Southern blot and RAPD analysis were performed to confirm that the insertion had affected only a single locus and that consequently the loss of the corresponding genetic function had led to a consistent reduction of virulence. A RAPD amplification product was found to be associated with the genomic region tagged by the plasmid, perhaps therefore representing a part of the inactivated gene. The accumulation of four defence-related transcripts was monitored in the root tips of cv. Nudinka challenged with both the virulent isolate and the isogenic hypovirulent T2. Two kinds of response were observed in rootlets inoculated with T2 when compared with the wild type: a delay in the accumulation of the mRNAs homologous to thaumatin-like protein, thionin and peroxidase genes and, 24 h after inoculation, a higher accumulation of mRNAs homologous to β-(1,3)-glucanases and thionins. The relationships between the plant defence response and the virulent and hypovirulent effects, as well as the potential use of this insertional mutant in the isolation and characterization of the inactivated virulence gene are discussed.