1. The in vitro effect of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) on mtDNA synthesis was studied using isolated newborn rat liver mitochondria. 2. From the kinetics of the incorporation of [3H]thymidine into the acid-insoluble material, MNNG neither stimulated nor inhibited the DNA synthesizing activity of mitochondria. The activity observed in the presence of MNNG was inhibited by N-ethylmaleimide and actinomycin D. 3. By the band velocity sedimentation in CsCl/ethidium bromide, the properties of the nascent mtDNA formed in the presence of MNNG were analyzed. The nascent DNA-containing molecule was not found in the closed-circle fraction, and essentially detected in the open-circle fraction. This change of the template was blocked by N-ethylmaleimide but not by actinomycin D, suggesting a conversion of the closed-circular template to the open-circular one by single-strand cleavage(s). From the band sedimentation in alkaline CsCl, the number of nascent higher molecular DNAs was increased but the molecules were all of relatively lower molecular weight. On the other hand, the formation of nascent fragments was inhibited. 4. The alkaline CsCl equilibrium centrifugation analysis revealed that the nascent DNA synthesized in the presence of MNNG consisted of both light and heavy components. 5. Present results suggest that MNNG exerts its effect on the mtDNA synthesis by modifying the intrinsic mechanism of discontinuous synthesis, since the conversion of the template DNA molecule from the closed- to open-circular form and the continuous polymerization of the nascent higher-molecular DNA on such a relaxed template were characteristic events in vivo.