In this paper we report on the investigation, as DNA nonviral carriers, of three samples of an amphoteric polyamidoamine bearing 4-aminobutylguanidine deriving units, AGMA5, AGMA10, and AGMA20, characterized by different molecular weights (M(w) 5100, 10100, and 20500, respectively). All samples condensed DNA in spherical, positively charged nanoparticles and protected it against enzymatic degradation. AGMA10 and AGMA20 polyplexes had average diameters lower than 100 nm. AGMA5 polyplexes were larger. All polyplexes showed negligible cytotoxicity and were internalized in cells. AGMA10 and AGMA20 performed differently from AGMA5 as nucleic acid carriers in vitro. AGMA10 and AGMA20 effectively promoted transfection, whereas AGMA5 was ineffective. FITC-labeled AGMA10 was prepared and the intracellular trafficking of its DNA polyplex was studied. DNA/AGMA10 polyplex was largely localized inside the nucleus, while AGMA10 concentrated in the perinuclear region. DNA/AGMA10 polyplex intravenously administered to mice promoted gene expression in liver but not in other organs without detectable toxic side effects.