Abstract Mitochondrial DNA was extracted from DNase I-treated mitochondrial preparations from a homokaryotic strain of the woodrotting basidiomycete Schizophyllum commune. An AT-rich satellite DNA was also isolated from both homokaryotic and dikaryotic strains using CsCl-bisbenzimide gradients. The satellite DNA and the DNA from DNase I-treated preparations of mitochondria have identical buoyant densities and hybridize to the same restriction fragments in Southern hybridizations. It is concluded that this satellite DNA is mitochondrial DNA. Restriction analyses of the mitochondrial DNA of strain 4–40 with five endonucleases show the mitochondrial genome to be about 49.85 kbp. Mitochondrial DNA from the 4–39 × 4–40 dikaryon has identical restriction fragments. The satellite, which we have shown to be mitochondrial DNA, represents 3–4% of the total cellular DNA and averages 22–29 copies per haploid cell under our growth conditions. Mitochondrial DNA prepared from three other strains of Schizophyllum revealed restriction enzyme polymorphisms and ranged in size from 50.3 to 52.2 kbp.