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 ∼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–PEG 8000 (5% ionic) nanoparticles were found to be the most efficient transfecting agent for in vitro transfection.