BACKGROUND Although polycations are among the most efficient nonviral vectors for gene transfer, the gene expression they allow is still too low for in vivo applications. To engineer more potent polycationic vectors, the factors governing the intracellular trafficking of a plasmid complexed with current polycations need to be identified. METHODS AND RESULTS The trafficking of plasmid DNA complexed to glycosylated polylysines or polyethylenimine (PEI) derivatives was studied by electron microscopy of human airway epithelial cells. The cellular processing of complexes varied with their size and the polycation derivative used: large complexes (> 200 nm) made with all polycationic vectors studied were internalized by macropinocytosis. In contrast, intermediate (100-200 nm) ligand-coupled polylysine and PEI complexes primarily entered through clathrin-coated pits. Complexes were then found in endosomal vesicles, accumulated in lysosomes or vesicles near the nucleus and their nuclear entry was limited. For the population of small complexes (< or = 100 nm) obtained with PEI derivatives, they were internalized through caveolae and pursued a traffic pattern of potocytosis to the endoplasmic reticulum where their fate remains unclear. Finally, some complexes exited the cells either by regurgitation when PEI derivatives were used or through an exosome-like pathway for glycosylated-polylysine complexes. CONCLUSIONS The different pathways of complex trafficking observed in relation with complex size imply the development and study of vectors forming complexes with definite size. Moreover, the complex exit we describe may contribute to the well-established short-term efficiency of gene transfer based on synthetic vectors. It favors the engineering of vectors allowing repeated treatment.