Polymeric nanoparticles of chitosan crosslinked with glutaraldehyde have been prepared using reverse micellar system. An optically clear solution was obtained on redispersing these nanoparticles in aqueous buffer. The nanoparticles were characterized for their size and surface morphology employing dynamic laser scattering (DES) and transmission electron microscopy (TEM). The TEM images showed spherical particles with smooth surface and narrow size distribution of about 90 nm, which was also supported by DLS data. Size and morphology of the particles remains the same on redispersing the lyophilized powder of these nanoparticles in aqueous buffer. Further, these nanoparticles were loaded with different synthetic oligonucleotides (ODNs). In vitro pH dependent release of the adsorbed oligonucleotides from these nanoparticles was also studied. At basic pH the release of oligonucleotides was found higher as compared with neutral and acidic medium. Cytotoxicity studies done on HEK 293 cells reveals that oligonucleotide loaded nanoparticles have high cell viability of nearly 76-88% whereas those of lipofectamine was about 35%. (C) 2010 Wiley Periodicals, Inc. J Appl Polym Sci 118: 2071-2077, 2010