Senescence in the filamentous fungus Podospora anserina is characterized by the accumulation of multimeric circular mitochondrial DNA molecules, known as senDNAs. These tandemly repeated DNA sequences, which originate from broadly dispersed regions of the young mitochondrial genome, behave as independently replicating molecules. In this study, the yeast transformation system was used to assay senDNAs and their young mtDNA counterparts for the presence of autonomously replicating sequences. P. anserina mtDNA fragments were cloned into the yeast vector YIp5 and the hybrid YPM plasmids were used to transform yeast. All of the senDNAs and their homologous young mtDNAs promoted high frequency transformation and extrachromosomal maintenance of YPM plasmids. The putative origin of replication for the P. anserina mitochondrial genome was also cloned into YIp5 and shown to confer autonomously replicating properties.