A series of well-defined amphiphilic triblock copolymers [polyethylene glycol monomethyl ether]-block-poly(epsilon-caprolactone)-block-poly[2-(dimethylamino)ethy l methacrylate] (mPEG-b-PCL-b-PDMAEMA or abbreviated as mPEG-b-PCL-b-PDMA) were prepared by a combination of ring-opening polymerization and atom transfer radical polymerization. The chemical structures and compositions of these copolymers have been characterized by Fourier transform infrared spectroscopy, H-1 NMR, and thermogravimetric analysis. The molecular weights of the triblock copolymers were obtained by calculating from H-1 NMR spectra and gel permeation chromatography measurements. Subsequently, the self-assembly behavior of these copolymers was investigated by fluorescence probe method and transmission electron microscopy, which indicated that these amphiphilic triblock copolymers possess distinct pH-dependent critical aggregation concentrations and can self-assemble into micelles or vesicles in PBS buffer solution, depending on the length of PDMA in the copolymer. Agarose gel retardation assays demonstrated that these cationic nanoparticles can effectively condense plasmid DNA. Cell toxicity tests indicated that these triblock copolymers displayed lower cytotoxicity than that of branched polyethylenimine with molecular weight of 25 kDa. In addition, in vitro release of Naproxen from these nanoparticles in pH buffer solutions was conducted, demonstrating that higher PCL content would result in the higher drug loading content and lower release rate. These biodegradable and biocompatible cationic copolymers have potential applications in drug and gene delivery. (C) 2010 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 48: 1079-1091, 2010