Ascospores from the phytopathogenic fungus Sclerotinia sclerotiorum were transformed to hygromycin B resistance by co-cultivation with Agrobacterium tumefaciens. Transformed spores germinated and grew on PDA supplemented with 100 ug/ml hygromycin B. The presence of mitotically stable hph gene integration at random sites in the genome was confirmed by PCR and Southern blot analysis. A transformation frequency of 8 x 10(-5) was achieved in five separate experiments. This study is the first report of success co-cultivating A. tumefaciens with S. sclerotiorum. This report of a reproducible Agrobacterium-mediated transformation method should allow the development of T-DNA tagging as a system for insertional mutagenesis in S. sclerotiorum and provide a simple and reliable method for genetic manipulation.