Copolymers consisting of branched PEI 25 kDa grafted with high molecular weight PEG at a low degree of substitution were successfully synthesized using a simple two-step procedure. The resulting AB-type and ABA-type copolymers were tested for cytotoxicity and DNA condensation and complexation properties. Their polyplexes with plasmid DNA were characterized in terms of DNA size and surface charge, transfection efficiency and blood compatibility. Pharmacokinetic profiles of the complexes containing (32)P-labeled plasmid were assessed before and after surface crosslinking. A set of four copolymers containing one or two PEG 20 kDa or PEG 30 kDa chains was obtained. The cytotoxicity of PEI was strongly reduced after copolymerization. The copolymer polyplexes showed hydrodynamic diameters of less than 200 nm, comparable to PEI 25. Similarly, no reduction in DNA condensation and complexation properties was found. In fact, PEI-PEG(30 k) copolymers exhibited better condensation and complexation properties than PEI 25. The transfection efficiency of copolymer polyplexes was increased 10-fold compared to PEI 25 control and the hemolytic activity was markedly reduced. After intravenous injection into mice, plasmids complexed to PEI-PEG(30 k) copolymers resulted in significantly increased circulation times. After stabilizing the polyplexes with a redox sensitive, biodegradable crosslinker, blood levels of plasmid could be further increased up to 125% compared to PEI. These results demonstrate that polyplexes prepared using a combined strategy of surface crosslinking and PEGylation seem to provide promising properties as stable, long circulating vectors.