Among many processes involved in nonviral gene delivery, intracellular fates of gene carriers are of great interest. Unlike viral vectors, which have been evolved to maximize intracellular delivery processes, inefficient and non-selective intracellular transport of synthetic gene carriers (e.g., polymer/DNA polyplexes) is believed as one of the major reasons for poor gene delivery efficiency. Utilizing various localization signals (e.g., NLS peptides) has been a major approach to overcome this challenge. In this study, targeted localization of synthetic polyplexes in the cytoplasm and the nucleus was explored by tailoring structures of a carrier polymer. It was also surmised that controlled types of therapeutic effects could be achieved by selectively localizing siRNA and plasmid DNA-containing polyplexes in their different intracellular destinations. A commonly used cationic polymer, polyethylenimine (PEI), was ketalized to increase cytosolic release but reduce cytotoxicity of polyplexes, while superior complexation and buffering capacity of the polymer were retained. Ketalized PEI (K-PEI) with different molecular weights at various ketalization ratios was synthesized. Acid-degradability, capability of nucleic acid condensation, and transfection and RNA interference efficiency of K-PEI/nucleic acid polyplexes were evaluated. It was clear that both siRNA and plasmid DNA condensed by K-PEI were efficiently released upon hydrolysis of ketal branches at endosomal pH. Almost completely diminished cytotoxicity of K-PEI/nucleic acid polyplexes was also demonstrated, regardless of molecular weights and N/P ratios. Surprisingly, polyplexes prepared with K-PEI of molecular weight of 0.8kDa transfected cells even more efficiently than 25kDa unketalized PEI, while there was no evidence of tranfection by unketalized 0.8kDa PEI polyplexes. This implies a possibility of using low molecular weight K-PEI for clinical nonviral gene therapy by utilizing its high transfection efficiency, low cytotoxicity, and eventual secretion from the body. More importantly, transfection efficiency of K-PEI polyplexes at the same ketalization ratio (i.e., 35%) was found to be inversely proportional to molecular weights of K-PEI, while higher RNA interference was observed with larger ketalized polymers. Further studies were carried to differentially control intracellular localization of siRNA and plasmid DNA-containing polyplexes by precisely modulating ketalization ratios of K-PEI in the range of 17-96%. Nucleic acid condensation, transfection efficiency, and RNA interference were found to be strongly correlated with ketalization ratios and molecular weights of K-PEI. On the contrary, it was proved that the the minimal ketalization (17%) was sufficient enough to diminish cytotoxicity of the polymer. The presentation will convey 1) logics of polymer design and synthesis, 2) preparation and characterization of K-PEI polyplexes using TEM, dynamic light scattering (DLS) analysis, and gel electrophoresis, 3) cytotoxicity test mainly by MTT assay, 4) results of transfection and RNA interference, and 5) evidence of selective localization of various K-PEI polyplexes by intensive confocal microscopic studies. Finally, implications of the study in achieving potent, biocompatible, and controlled nonviral gene delivery will be discussed.