Ditercalinium chloride was originally synthesized for use as an anticancer drug and was then found to deplete mitochondrial DNA. Ethidium bromide is widely used to deplete mitochondrial DNA and produce mitochondrial DNA-less cell lines. Although ethidium bromide is used in the case of human cell lines, it frequently fails to deplete mitochondrial DNA in mouse cells. In contrast, ditercalinium chloride can deplete mitochondrial DNA in both mouse and human cells. However, little is known of the mechanisms by which ditercalinium chloride depletes mitochondrial DNA. Here, we show that ditercalinium chloride inhibits human DNA polymerase gamma activity as efficiently as does ethidium bromide. Ethidium bromide accumulates much less in mouse B82 cells, as compared with findings in human HeLa cells, whereas ditercalinium chloride accumulates in both to a similar extent. This poor accumulation of ethidium bromide may, in part, account for the resistance. Ethidium bromide distributes diffusely in the mitochondria of HeLa cells, while ditercalinium chloride distributes granularly and hence may be strongly associated with mitochondrial DNA. Each granular spot presumably represents one mitochondrial DNA nucleoid. In support of this idea, ditercalinium chloride co-localizes with Twinkle, a mitochondrial helicase and is assumed to associate with mitochondrial DNA. This close association of ditercalinium chloride with mitochondrial DNA may contribute to the mitochondrial DNA-depleting activity.