Gene therapy strategies depend on efficient devices for the delivery of nucleic acid into target cells. In this study, DNA-chitosan microspheres were investigated from a pharmaceutical standpoint and the effects of different factors such as plasmid size, chitosan concentration and plasmid addition techniques, on the characterization and in vivo transfection of microspheres were studied Different doses of DNA-chitosan microspheres were also tested for their gene expression properties. Large and small plasmids (pMK3 and pUC18, respectively) were used to study the importance of plasmid size to in vitro DNA release and transfection properties. Naked and encapsulated plasmids were injected into the muscle of the rats and their beta-galactosidase production was measured. According to our data, the effect of plasmid size and chitosan concentration on transfection efficacy was not clear. However, the DNA dose injected in rats was of importance to beta-galactosidase production. The highest levels of beta-galactosidase expression were obtained with low-dose DNA-chitosan microspheres. The injection of naked plasmid DNA to rats resulted in negligible expression. On the other hand the structural and functional integrity of encapsulated plasmid DNA remained unchanged during the study.