In nonviral transfection systems, the gene carrier/DNA complex must undergo several steps for successful transgene expression. DNA release from the complex is one important step. However, the detailed mechanism of intracellular processes involved in DNA release is not well understood. In this study, to clarify the dissociation of the complex in the cytosol, we investigated whether the DNA release was caused by addition of a cytosolic fraction prepared from mouse liver to the complex. When Lipofectamine (a liposome-type gene carrier) was used as a complex forming reagent with DNA, the cytosolic fraction caused no DNA release from the complex. In contrast, when dendritic poly(L-lysine) and jetPEI (polymer-type gene carriers) were used, DNA release was observed when the complex formed at a low cation/anion ratio. This result showed that a DNA releasing factor was present in the cytosolic fraction, suggesting that in the cytosol the DNA was spontaneously released from a gene carrier/DNA complex when the carrier was a polymer-type gene carrier. Furthermore, this DNA releasing ability of the cytosolic fraction was protease sensitive.