Abstract We succeeded in visualizing plasmid DNA (pDNA) in the nucleus and cytosol of non-proliferative cells after transfection with linear polyethylenemine (lPEI) and histidinylated lPEI (His16-lPEI). This was possible with confocal microscope by using pDNA labelled with quantum dots. Indeed pDNA labelled with Cy3 leads to false positive nuclear localization because the saturation of the fluorescence signal overestimated the volume occupied by Cy3-pDNA. Moreover, Cy3 brightness was too weak to detect low amount of pDNA. About 20 to 40 pDNA copies were detected in the nucleus after the transfection of pDNA labelled with quantum dots. Transfection efficiency and cellular imaging data suggested that the cytosolic availability of pDNA, including endosome escape and/or polyplexes dissociation, is crucial for its nuclear delivery. In vitro transcription assay and transfection of cells allowing cytosolic gene expression concluded to better cytosolic availability of pDNA within His16-lPEI polyplexes. Cryo-TEM analyses revealed that His16-lPEI polyplexes exhibited a spherical shape and an amorphous internal structure which differed from the high degree of order of lPEI polyplexes. Altogether, this comparative study indicated that the high transfection efficiency of non-proliferative cells with His16-lPEI polyplexes was related to the amorphous structure and the facilitated dissociation of the assemblies.