The molecular weight (MW) of chitosan (CS) was determined by viscometric method (using Mark-Houwink equation) as well as by gel permeation chromatography, and the degree of deacetylation (DDA) of CS was measured by potentiometric titration method and Gran-type linearization method. The values of DDA were obtained similar to 83% (by potentiometric titration method) and similar to 86% (by Gran-type linearization method). The self-assembled nanoparticles of CS/plasmid DNA (pDNA) complex were prepared by varying the concentration of CS. The formation of CS/pDNA complex was confirmed by 0.8% agarose gel electrophoresis and the particle size of the self-assembled nanoparticle was determined by dynamic light scattering, atomic force microscopy and scanning electron microscopy. The stability of CS/pDNA complexes was determined by turbidity test with the help of UV-Vis spectroscopy. The effect of ionic strength on the complexes was also observed by means of fluorescence spectroscopy. The cytotoxicity of CS on the HeLa cell line was observed by absorbance of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and showed that CS has lower cytotoxicity in HeLa cells compared with that of poly (L-lysine) in 293T cells. (C) 2011 Wiley Periodicals, Inc. J Appl Polym Sci 121: 2239-2249, 2011