In the maize pathogenic fungus Ustilago maydis integration of transforming DNA at homologous or heterologous sites is often accompanied by duplications of the DNA. We show that it is possible to generate single-copy integration events with high efficiency by restriction enzyme-mediated integration (REMI). In about 50% of cases, a plasmid that contains a single BamHI site is integrated at chromosomal BamH1 sites, if BamHI is added to the transformation mixtures. In the other cases it appears that integration events have also occurred preferentially at BamHI sites, but without restoration of the recognition sites. Using REMI we have generated approximately 1000 insertion mutants. Pathogenicity tests demonstrated that about 1-2% of these mutants were unable to induce symptoms when tested in planta. For two of the mutants we have shown that the phenotype is linked to the insertion event.