Diblock copolymers (MPEG-b-PCLs) of poly(epsilon-caprolactone) (PCL) and monomethoxyl poly(ethylene glycol) (MPEG) were synthesized by the conventional ring-opening polymerization of epsilon-caprolactone using MPEG as a macroinitiator. The monohydroxy-bearing diblock copolymers were reacted first with maleic anhydride and then with N-hydroxysuceinimide (NHS) to yield activated succinimidyl carbonate derivatives that are reactive with the primary amino group. Subsequently, a new class of biodegradable amphiphilic copolymer (hy-PEI-g-PCL-b-PEG) was prepared by grafting the activated PCL-b-PEG onto the hyperbranched poly(ethylene imine) (hy-PEI). Thermal properties of bulk graft copolymers were investigated using differential scanning calorimetry and thermogravinietric analysis. Depending on their compositions, these polymers are completely soluble in water or form micelles of tens to hundreds of nanometers in size in the studied concentration range, as revealed by surface tension and dynamic light scattering measurements of copolymer solutions. Complexation of plasmid DNA (pDNA) with various copolymers was investigated to achieve particles of ca. 200 nin diameter (N/P = 7). Copolymer composition was found to affect significantly the gene transfection efficiency of polyplexes. In general, low graft density and high molecular weight of PEI blocks favor high gene transfection efficiency. All DNA/copolymer complexes (N/P = 7) showed a much lower xi-potential (i.e., neutral or negative) than the DNA/PEI25 kDa complex (21 mV), indicating lower toxicity of copolymer-based complexes. Lower cytotoxicity of DNA/copolymer complexes was also demonstrated by the viability of cells in the transfection experiments. These results indicate that these ternary copolymers are promising candidates for gene delivery, featuring good biocompatibility, potential biodegradability, and relatively high gene transfection efficiency. Their neutral surface charge offers potential for intravenous administration.