A barbell-like ABA-type triblock copolymer, poly(L-lysine) dendrimer-block-poly(ethylene glycol) block-poly(L-lysine) dendrimer (PLLD-PEG-PLLD), was synthesized by the liquid-phase peptide synthesis method. The self-assembling complex formation of the third and fourth generation of the copolymer with plasmid DNA was studied. H-1 NMR and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) were used for the characterization of the synthesized copolymer. The self-assembling behavior of the 3rd and 4th generations of the copolymer with plasmid DNA was investigated by electrophoretic mobility shift assay, DNase I protection assay, and ethidium bromide exclusion assay. We observed great differences in the self-assembling ability of the 3rd and 4th generations of the polymer. This suggests that the number of positively charged amines per polymer molecule should be an important factor for the potential for self-assembling complex formation with DNA. Atomic force microscopy (AFM) and zeta potentials were used for evaluating the shape, size distribution, and surface charge of the complexes at various charge ratios. From AFM images, it was observed that the shape of the complex was nearly spherical and its size was about 50-150 nm in diameter. The in vitro cytotoxicity of the copolymer was compared with that of poly(L-lysine), poly(D-lysine), and polyethylenimine.