1) We have identified by electron microscopy and isolated a circular DNA species of approximately 6 µm contour length from DNase treated mitochondrial fractions of the petite negative yeast Schizosaccharomyces pombe (S. pombe). 2) Another molecular species of about 3 µm length is also present in mitochondrial fractions. These molecules, however, disappear after DNase treatment or extensive washing, indicating their extramitochondrial location. There is evidence (Fournier et al. 1981) that these molecules represent a multicopy plasmid coding for the genes of cytoplasmic ribosomal RNAs. 3) A restriction enzyme cleavage map of the 6 µm species was constructed using twelve enzymes. Physical mapping revealed a genome length of approximately 18.9 kilobase pairs, thus confirming the electron microscopic data. 4) Northern hybridization of mitochondrial RNA with restriction fragments of mitochondrial DNA revealed two major signals which are attributed to the small and large ribosomal RNA. Apparently both rRNA genes map close together. 5) Spontaneous mit(-) deletion mutants were characterized phenotypically and the location of their deletions was determined. 6) In strains carrying a cytoplasmic mutator (Seitz-Mayr and Wolf 1982) or derived from mutator strains four restriction sites have been mapped, which are not present in the mitochondrial DNA of the parental strain. These extra sites are very likely consequences of the action of the mutator. 7) In conclusion we have presented evidence that the 6 µm circular DNA species is the mitochondrial genome of fission yeast. Special features of this genome are discussed in comparison with other mitochondrial genomes. 