A transformation system utilizing Agrobacterium tumefaciens was developed for targeted gene disruption in Monilinia fructicola, a fungal pathogen that causes brown rot disease of stone fruits. Transformation with a vector containing the neomycin phosphotransferase II (nptII) cassette flanked with 4 kb cutinase gene (Mfcut1) flanking sequences resulted in an average of 13 transformants per 10(5) spores. When assayed by PCR and DNA blot analyses, more than 50% of the transformants recovered had integrated in the targeted Mfcut1 locus. Both target-gene-specific and non-specific integrations carried direct (head-to-tail) repeat T-DNA integrations. Sequence analysis of these T-DNA integrations revealed that 26 bp of the T-DNA right border were missing at the junctions between direct repeats in all cases. The recombination event during non-specific T-DNA integration in this fungus was unlike that reported in Agrobacterium-mediated transformation in plants.