We here addressed the basic question, how does extrachromosomal DNA behave when it is placed in the nuclear or the cytoplasmic environment and how is it eliminated? To do this, we tracked microinjected DNA molecules in live cells. In the cytoplasm, the diffusion of microinjected DNA was inhibited in a size- and linearity-dependent manner, probably by the intermediate filament. This was followed by the rapid disappearance of the DNA fluorescent signal. In the nucleus, the diffusion was also dependent on the size of the molecule and was accompanied by the aggregation of the DNA. The aggregation may be due to a putative DNA-binding molecule, whose level is high during the G1 phase. Surprisingly, the injected DNA could move across the nuclear membrane and appeared in the cytoplasm, which suggests the presence of a transport system. The intracytoplasmic behavior and the elimination of such DNA was obviously different from the DNA that was directly injected at the cytoplasm. The DNA remaining in the nucleus appeared to be stable and persisted in the nucleus or, after cell division, in the cytoplasm, for more than one cell cycle. These findings provide a novel and basic understanding of the behavior and elimination of a wide variety of extrachromosomal genetic material.