Pyrenophora tritici-repentis causes tan spot, an important foliar disease of wheat. The fungus produces multiple host-specific toxins, including Ptr ToxB, a chlorosis-inducing protein encoded by the ToxB gene. A homolog of ToxB is also found in avirulent isolates of the fungus. In order to improve understanding of the role of this homolog and evaluate the general pathogenic ability of P. tritici-repentis, we compared the proteomes of avirulent race 4 and virulent race 5 isolates of the pathogen. Western blotting analysis revealed the presence of Ptr ToxB in spore germination and culture fluids of race 5 but not race 4. A comprehensive proteome-level comparison by 2-DE indicated 133 differentially abundant proteins in the secretome (29 proteins) and mycelium (104 proteins) of races 4 and 5, of which 63 were identified by MS/MS. A number of the proteins found to be up-regulated in race 5 have been implicated in microbial virulence in other pathosystems, and included the secreted enzymes alpha-mannosidase and exo-beta-1,3-glucanase, heat-shock and BiP proteins, and various metabolic enzymes. These proteome-level differences suggest a reduced general pathogenic ability in race 4 of P. tritici-repentis, irrespective of toxin production. Such differences may reflect an adaptation to a saprophytic habit.