Sustained release polymeric gene delivery systems offer increased resistance to nuclease degradation, increased amounts of plasmid DNA (pDNA) uptake, and the possibility of control in dosing and sustained duration of pDNA administration. Furthermore, such a system lacks the inherent problems associated with viral vectors. Biodegradable and biocompatible poly(DL-lactide-co-glycolide) polymer was used to enacapsulate pDNA (alkaline phosphatase, AP, a reporter gene) in submicron size particles. Gene expression mediated by the nanoparticles (NP) was evaluated in vitro and in vivo in comparison to cationic-liposome delivery. Nano size range (600 nm) pDNA-loaded in poly(DL-lactide-co-glycolide) polymer particles with high encapsulation efficiency (70%) were formulated, exhibiting sustained release of pDNA of over a month. The entrapped plasmid maintained its structural and functional integrity. In vitro transfection by pDNA-NP resulted in significantly higher expression levels in comparison to naked pDNA. Furthermore, AP levels increased when the transfection time was extended, indicating sustained activity of pDNA. However, gene expression was significantly lower in comparison with standard liposomal transfection. Seven days after i.m. injections in rats, naked pDNA and pDNA-NP were found to be significantly more potent (1-2 orders of magnitude) than liposomal pDNA. Plasmid DNA-NP treatment exhibited increased AP expression after 7 and 28 days indicating sustained activity of the NP.