Mitochondrial nucleoids (mt-nucleoids) were isolated from 6 species of yeasts that belong to Saccharomyces cerevisiae, S. dairenensis, S. kluyveri, S. servazzii, S. exiguus, and Arxiozyma telluris. Each fraction during the mt-nucleoid isolation was analyzed for the detection of Abf2p-like DNA-binding protein and mitochondrial nuclease by SDS-DNA PAGE. This method revealed that the unique Abf2p-like protein, which ranges in molecular mass from 18—21 kDa, is associated with each mt-nucleoid fraction isolated from 6 yeasts. The appearance of nuclease activity on SDS-DNA gels significantly differed depending on the yeasts tested, and the nuclease activity in the mitochondrial fraction was detected in only 2 species of yeasts, S. kluyveri and A. telluris. In contrast, the nuclease activity was detected in the mt-nucleoid fraction, but not the mitochondrial fraction, of S. cerevisiae and S. exiguus. The zymographic assay on SDS-DNA gels showed that the mitochondrial nuclease from S. kluyveri and A. telluris was activated by Mn2+, Mg2+, Ca2+, but not by Zn2+. The effects of Co2+ on the mitochondrial nuclease activity differed between the 2 yeasts.