Tan spot, caused by the fungus Pyrenophora tritici-repentis, is an important foliar disease of wheat. The pathogen produces at least three host-specific toxins, including Ptr ToxB, a 6·6 kDa protein that causes chlorosis in sensitive wheat genotypes and is encoded by the ToxB gene. Using quantitative reverse transcriptase (qRT)-PCR, transcript levels of ToxB homologues found in virulent (≡ pathogenic) and weakly virulent (≡ weakly pathogenic) race 5 isolates and an avirulent (≡ nonpathogenic) race 4 isolate of the fungus were compared in conidia, mycelia, and in host tissues after inoculation of resistant and susceptible wheat genotypes. Abundance of the ToxB transcript was greatest in the virulent isolate, followed by the weakly virulent and avirulent isolates, and was positively correlated with the development of chlorosis in the susceptible wheat genotype; transcript was detectable in mycelia and conidia of all isolates tested, and levels peaked at 24 h after inoculation of the race 5 isolates onto resistant and susceptible wheat hosts, before disease symptoms developed. Although all isolates could penetrate host epidermal cells to varying degrees, transcript abundance was positively correlated with the greater and more rapid development of appressoria. Therefore Ptr ToxB may have other roles in the basic pathogenic fitness of the fungus and/or in pre-penetration processes, suggesting that quantitative variation in the virulence of P. tritici-repentis is related to the extent of ToxB gene expression.