Polyethylenimine (PEI) is an efficient vector for in vitro and in vivo gene transfer into respiratory cells. Glycosylated PEIs were shown to enhance in vitro gene transfer by favoring the complex entry into the airway cells. The aim of our study was to evaluate the in vivo efficiency of gene transfer mediated by glycosylated PEIs in the mouse lung and to determine the transfected cell type and the intracellular trafficking of the complexes. Upon nasal instillation in mice of complexes made with various glycosylated PEIs, a high luciferase activity was observed while the green fluorescent protein (GFP) expression was similar for all the vectors tested with few cells expressing GFP. Complexes made with lactosylated PEI were then labeled and their localization studied by confocal microscopy. In the lungs, large numbers of complexes were taken up by epithelial cell which were mostly alveolar cells. In the airways, complex uptake varied greatly, depending on the area observed. Eight hours upon nasal instillation and in contrast with the in vitro situation, a dissociation between the plasmid DNA and the lactosylated PEI was usually observed, leading to the plasmid mostly localized in lysosomes and the Lac-PEI localized in the nucleus. These results emphasize the need to engineer a plasmid able by itself to overcome the nuclear barrier and to quickly move to in vivo experiments to select the best carrier.