Objective: Interaction of cell-surface glycosaminoglycans (GAGs) with nonviral vectors seems to be an important factor which modifies the intracellular destination of the gene complexes. Intracellular kinetics of polyamidoamine (PAMAM) dendrimer as a nonviral vector in intranuclear delivery of DNA, transcription and translation steps with regard to the cell-surface GAGs has not been investigated until now. Materials and Methods: The physicochemical properties of the PAMAM-DNA complexes were characterized by photon correlation spectroscopy, atomic force microscopy, zeta measurement and agarose gel electrophoresis. The transfection efficiency and toxicity of the complexes at different nitrogen to phosphate (N: P) ratios were determined using various in vitro cell models (human embryonic kidney cells, chinese hamster ovary cells and its mutants lacking cell-surface GAGs). Intranuclear uptake, transcription and translation expression of the particles were determined using optimized cellnuclei isolation with quantitative real-time PCR and luciferase assay. Results: Physicochemical studies showed that PAMAM G5-DNA binds DNA efficiently, forms small complexes with high positive zeta potential and transfects cells properly at N:P ratios around 5 and higher. The cytotoxicity could be a problem at N: Ps higher than 10. GAGs elimination caused nearly one order of magnitude higher DNA nuclear uptake and more than 2.6-fold higher transgene expression than CHO wild-type cells. However, neither AUC of nuclear uptake, nor AUC of transgene expression affected significantly by only cell-surface heparin sulphate proteoglycans (HSPGs) elimination and interesting data related to the effect of GAGs on intranuclear pDNA using PAMAM as delivery vector have been reported in this study. Conclusion: Presented data shows that the rate-limiting step of PAMAM-DNA particle transfection is located after delivery to the cell nucleus and GAGs are regarded as an inhibitor of the intranuclear delivery step, while slightly promotes transcription or translation process.