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Artificial nonviral gene vectors have the potential to improve the safety of gene therapy; however, such artificial vectors are less efficient for gene expression due to the existence of various barriers to delivery of exogenous DNAs to the nucleus in the living cell. Here we describe the degradation activities of cytoplasmic nucleases, which are involved as a barrier to efficient exogenous gene expression. The size and structure of degraded DNA were monitored by fluorescence correlation spectroscopy (FCS) and fluorescence cross-correlation spectroscopy (FCCS) in solution and in the cytoplasm of living cells. Differences in degradation by endo- and exonucleases were confirmed by differences in the size and structure of DNA. Moreover, we confirmed the influence of the exonuclease degradation in cytoplasm on the expression rate of DNA transfection with a cationic lipid. Based on a comparison of in vitro and cell measurements, we conclude that cytoplasmic degradation by exonucleases can be a considerable barrier against efficient gene delivery.Our method using noninvasive monitoring of the diffusion properties of DNA provides information on the fate of intracellular exogenous DNA and the efficiency of DNA integration in living cells.