Parental administration of polyplex formulations for gene therapy or genetic vaccination requires sterile preparations. The possibilities and limitations of autoclaving, filtration and a combination of both methods for sterilization of poly(2-(dimethylamino) ethyl methacrylate) (pDMAEMA) based gene transfer complexes were assessed. Agarose gel electrophoresis and circular dichroism spectroscopy showed that sterile filtration of polyplexes did not change the topology and integrity of the DNA. The transfection potential was fully retained in COS-7 and OVCAR-3 cells, although the concentration of DNA was slightly decreased by the filtration process. Pre-coating of the filter with polyplexes reduced the material loss. In contrast, autoclaving dramatically affected physical characteristics of polyplexes, resulting in complete loss of transfection potential. Sterile filtration or autoclaving of polymer alone did not result in material loss, or in decreased transfection potential after complexation with plasmid DNA. 'Naked' DNA could easily be sterilized by filtration as well. In conclusion, sterilization of complexes between pDMAEMA-based cationic polymeric gene transfer agents and DNA plasmid is feasible by filtration. Depending on the filter type used, the filtered volume should be high enough, to prevent substantial material loss. Separate sterilization of the polymer by autoclaving or filtration and DNA by filtration offers a good alternative to filtration of formed polyplexes.