Mitochondrial nucleoids (mt-nucleoids) isolated from the yeast Saccharomyces cerevisiae were analyzed to identify the protein components that are involved in the compact packaging of mtDNA. The isolated mt-nucleoids were disassembled by the addition of 2 M NaCl and the disassembled mt-nucleoids were reassembled once again into compact structures by dialysis against a buffer that contained NaCl at concentrations below 0.1 M, as monitored by staining of the DNA with 4',6-diamidino-2-phenylindole. DNA-binding proteins with molecular masses of 67 kDa, 52 kDa, 50 kDa, 38 kDa, 30 kDa and 20 kDa were separated from isolated mt-nucleoids by column chromatography on DNA-cellulose after digestion of mt-nucleoids by DNase I in the presence or absence of 2 M NaCl. Purified mtDNA was compactly packaged into nucleoid-like structures upon the addition of fractions that contained DNA-binding proteins and subsequent dialysis to reduce the concentration of NaCl. Five proteins, with molecular masses of 67 kDa, 52 kDa, 50 kDa, 38 kDa and 30 kDa, respectively, had lower affinity for double-stranded DNA than that of the 20-kDa protein. The fraction that contained the five DNA-binding proteins other than the 20-kDa protein was also able to fold mtDNA compactly into nucleoid-like structures. By contrast, the combination of the 20-kDa protein and mtDNA resulted in formation of less tightly packed, string-of-bead structures. These results suggest that at least six different DNA-binding proteins are involved in the organization of the mt-nucleoids.