Fusarium rot caused by Fusarium oxysporum f.sp. gladioli (Fog) is one of the most serious diseases of Gladiolus, both in the field and in bulbs in storage. In order to study the mechanisms of pathogenesis of this fungus, we have transformed Fog with Agrobacterium tumefaciens binary vectors containing the hygromycin B phosphotransferase (hph) gene and fluorescence reporter genes EGFP (green), EYFP (yellow) or ECFP (cyan) using the AGL-1 strain of A. tumefaciens. Hygromycin B (100 μg/ml) resistant colonies were observed only when acetosyringone was added to the co-cultivation medium. Transformed colonies are more clearly visible when co-cultivated on cellophane membrane than on Hybond -N+ membrane. Transformed lines were stably maintained through four serial passages on medium containing hygromycin B, and they expressed green, yellow or cyano fluorescence. PCR with hph-specific primers and Southern blotting with an hph-specific probe were positive for HygR lines but not for the untransformed isolate. The cyano fluorescence of the ECFP-transformed isolate was clearly distinguishable from the green autofluorescence of Gladiolus roots, signifying the potential of these lines for further histopathological investigations. Transformed lines will be useful for identifying pathogenicity related genes, screening transgenic resistance, and in studies of host-pathogen interactions.