Local and controlled DNA release is a critical issue in current gene therapy. As viral gene delivery systems are associated with severe security problems, nonviral gene delivery vehicles were developed. Here, DNA-nanoparticles using grafted copolymers of PLL and PEG to increase their biocompatibility and stealth properties were systematically studied. Ten different PLL-based polymers with no, low, and high PEG grafting and PEG molecular weights as well as different PLL backbone lengths were complexed with plasmids containing 3200 to 10,100 base pairs. Stable complexes were formed and selected for cytotoxicity and transfection efficiency. Predominantly, PLL-g-PEG-DNA nanoparticles grafted with 4 or 5% PEG moieties of 5 kDa transfected 40% COS-7 cells without reduction of cell viability when formed at N/P ratios between 0.1 and 12.5. The molecular weight of PLL did not significantly affect transfection efficiency or cytotoxicity indicating that a specific cationic charge-density-to-PEG-ratio is important for efficient transfection and low cytotoxicity. The PLL-g-PEG-DNA nanoparticles were spherical with a diameter of approximately 100 nm and did not aggregate over 2 weeks. Moreover, they protected included plasmid DNA against serum components and DNase I digestion. Therefore, such storage stable and versatile PLL-g-PEG-DNA nanoparticles might be useful to deliver differently sized therapeutic DNA for in vivo applications.