The necrotrophic fungus Pyrenophora tritici-repentis is responsible for the disease tan spot of wheat. Ptr ToxB (ToxB), a proteinaceous host-selective toxin, is one of the effectors secreted by P. tritici-repentis. ToxB induces chlorosis in toxin-sensitive wheat cultivars and displays characteristics common to apoplastic effectors. We addressed the hypothesis that ToxB exerts its activity extracellularly. Our data indicate that hydraulic pressure applied in the apoplast following ToxB infiltration can displace ToxB-induced symptoms. In addition, treatment with a proteolytic cocktail following toxin infiltration results in reduction of symptom development and indicates that ToxB requires at least 8 h in planta to induce maximum symptom development. In vitro assays demonstrate that apoplastic fluids extracted from toxin-sensitive and -insensitive wheat cultivars cannot degrade ToxB. Additionally, ToxB can be reisolated from apoplastic fluid after toxin infiltration. Furthermore, localization studies of fluorescently labeled ToxB indicate that the toxin remains in the apoplast in toxin-sensitive and -insensitive wheat cultivars. Our findings support the hypothesis that ToxB acts as an extracellular effector.