Two cross-linkers based on polyethylene glycol (PEG) (MW=6 and 8 kDa), were synthesized for self-assembling and formation of nanoparticles of branched, high molecular weight polyethylenimine (PEI). Cross-linking was realized in two ways, viz., ionic as well as covalent. Ionic cross-linking was accomplished by using PEG-bis (phosphate) whereas, the covalent one was achieved by using PEG-bis (p-nitrophenylcarbonate). A range of nanoparticles of PEI was prepared by varying the degree of cross-linking (i.e. the amount of cross-linkers used). PEI-PEG nanoparticles were characterized by dynamic light scattering and transmission electron microscopy and found to be in the range of approximately 18-75 nm (hydrodynamic radii) with almost uniform population. Subsequently, these particles were used for DNA binding assay and zeta-potential measurements, taking native PEI-PEG nanoparticles as reference. As expected, the zeta potential values decreased, on increasing the percentage of cross-linking as well as on complexation with DNA. Further, PEI-PEG nanoparticles were investigated for their transfecting efficacy on COS-1 cells. It was found that PEI-PEG nanoparticles were 5- to 16-fold more efficient as transfecting agents compared to lipofectin and PEI itself. The toxicity of PEI-PEG nanoparticles was found to be reduced considerably in comparison to PEI polymer, as determined by MTT colorimetric assay. Out of the various systems prepared, PEI-PEG8000 (5% ionic) nanoparticles were found to be the most efficient transfecting agent for in vitro transfection.