The aim of this study is to evaluate transfection efficiency in NIH/3T3cell using DNA/polyethyleneimine (PEI) complexes. The formation of complexes from DNA and PEI was performed by the adding of the PEI solution to the DNA solution. Gel retardation assay showed the optimal N/P charge ratio where PEI completely binds DNA. The particle size of DNA/PEI complexes measured by dynamic light scattering showed diameters of up to 500 nm. The viability of NIH/3T3 fibroblast cells in the presence of DNA/PEI complexes was evaluated by the MTT assay. After gene transfection, enhanced green fluorescence protein was monitored by flow cytometry. From AFM measurement, DNA/PEI complexes showed round-shape. These results indicated that DNA was sufficiently condensed by PEI to give DNA/PEI complexes. As the relative amount of PEI was increased, its cytotoxicity to NIH/3T3cells increased. By increasing culture time, fluorescence microscopy showed a more green fluorescence cells. Naked DNA showed no EGFP expression, whereas delivery of DNA/PEI complexes to cells showed EGFP expression and resulted in 2-15% transfection. Transfection efficiencies of DNA/PEI complexes increased with increasing N/P charge ratio.