Fungi are lower eukaryotes that play important roles in many human activities, including biotechnological processes, phytopathology, and biomedical research. In addition, they are excellent models for molecular and genetic studies. An important key in the advancement of genetics and molecular biology of a given organism is the development of genetic transformation systems. This technology makes possible the analysis and manipulation of the genome of the organism of interest. Thirty years from the first report of transformation of a fungus, transformation of many other fungi has been achieved. However, the development of gene tagging systems generally applicable to a wide range of filamentous fungi has remained elusive. A widely used gene tagging strategy for filamentous fungi is restriction enzyme mediated integration (REMI). In recent years numerous reports have been published describing the effective application of REMI. However, REMI shows certain disadvantages for some fungi. Recently a very promising alternative strategy has been reported based on the use of the soil bacterium Agrobacterium tumefaciens. Using this system a well-defined DNA segment (T-DNA) is transferred, which integrates by illegitimate recombination and is 100–1000 times more efficient than conventional methods. The T-DNA can be used as an efficient tool to generate recombinant strains where DNA is integrated as a single copy, allowing the generation of collections of gene-tagged mutants of the fungus of interest.