We have developed in Podospora anserina a two-step procedure for DNA sequence replacement through transformation which might be applicable to other filamentous fungi. Targeting of transforming DNAs to their homologous locus is achieved provided a cosmid vector is used. Southern blot analysis of genomic DNAs from a set of transformants is presented. The data confirm that cosmids integrate into the chromosome through mostly homologous recombination which leads to a duplicated sequence separated by the vector. This event was found to be unstable in crosses. We show that this instability is due to the frequent excision of the vector together with the selective marker and one copy of the duplication, either the resident or foreign sequence. The two sequences can be distinguished because they exhibit restriction fragment length polymorphism. Therefore, Podospora anserina treats duplications occurring through transformation in a way differing from that exhibited by Neurospora crassa and Ascobolus immersus.