Two series of block copolymers were prepared by grafting linear poly(ethylene glycol) (PEG) onto branched polyethylenimine (PEI). In the first series, the PEI (25 000) was grafted with varied numbers of PEG blocks (5000). The second series was composed of copolymers all containing an equal amount of PEG (50%) and PEI (50%), but with PEG of different molecular weights (MW: 550-20 000). In a two-step synthesis, both the activation of monomethyl-PEGs and the coupling reactions of the PEGs with PEI were performed with hexamethylene diisocyanate (HMDI), leading to water-soluble copolymers with hydrolytically stable urethane and urea bonds. The molecular structure of the resulting copolymers was evaluated spectroscopically (NMR, FTIR). Thermal and calorimetric analysis (TGA, DSC) as well as size exclusion chromatography (SEC) verified the successful formation of the copolymers. MW was determined by static light scattering in combination with SEC. With respect to their application as gene transfer agents, the biocompatibility of the copolymers was studied using an in vitro cytotoxicity assay (lactate dehydrogenase assay) and blood compatibility tests (hemolysis and erythrocyte aggregation). It was found that PEG reduced the toxicity of PEI and prevented hemolysis as well as the aggregation of erythrocytes. The extent of the positive influence of PEG on the biocompatibility of the copolymers was found to be dependent upon both the number of PEG blocks and the structure of the block copolymers.