The molecular organization of mitochondrial-nucleoids (mt-nucleoids) isolated from BY-2 cultured tobacco (Nicotiana tabacum L.) cells was analyzed. Fluorescence microscopy using 4′,6-diamidino-2-phenylindole (DAPI) revealed that isolated mt-nucleoids retained the compact structural organization as that observed in vivo. Treatments of mt-nucleoids with DNase I, proteinase K and 2 M NaCl resulted in the disappearance, partial swelling, or complete dispersion of their DAPI-fluorescence, respectively, whereas treatment with RNase A did not affect their compact appearance. These results suggest that the compact structure of mt-nucleoids is maintained by electrostatic interaction between DNA and proteins. When mt-nucleoids that were treated with micrococcal nuclease (MNase) were analyzed by agarose gel electrophoresis, a ladder-like electrophoretic pattern of DNA, whose minimum fragment size was about 75 bp, was observed. This result suggests the presence of nucleosome-like, repetitive structures in the mt-nucleoids. Treatment of mt-nucleoids with increasing concentrations of NaCl gradually solubilized various proteins, including those with DNA-binding activities, which was accompanied by gradual dispersion of the mt-nucleoid structure. This result suggests that a variety of DNA-binding proteins with differing degrees of binding strength are present in the mt-nucleoids, and are involved in the compact organization of DNA-protein complexes at various levels. Detailed analyses of mt-nucleoids by Southwestern blotting, SDS-DNA PAGE, and DNA-cellulose affinity chromatography revealed the presence of many different DNA-binding proteins in the 2 M NaCl-soluble fraction of mt-nucleoids, which are expected to be involved in the compact organization of mt-nucleoids at various levels.