Agrobacterium tumefaciens-mediated transformation has been successfully applied to the wheat pathogen Mycosphaerella graminicola. Both protoplasts and intact cells have been transformed to hygromycin B resistance. Furthermore, A. tumefaciens-mediated transformation using homologous DNA originating from the M. graminicola ABC transporter gene MgAtr2 resulted in the efficient generation of disruption mutants. In 44% of the transformants, disruption of MgAtr2 was achieved and transformants resulted from the integration of a single copy of the transforming DNA. These results indicate that A. tumefaciens-mediated transformation is a useful tool to generate targeted gene disruption in the phytopathogen M. graminicola, where gene targeting by conventional methods is hardly possible.