RNA interference (RNAi) represents a powerful method for specific gene silencing. It is mediated through small double-stranded RNA molecules (small interfering RNAs, siRNAs) which sequence-specifically trigger the cleavage and subsequent degradation of their target mRNA. One critical factor that determines the success of RNAi is the ability to deliver intact siRNAs into target cells. Polyethylenimines (PEIs) are synthetic polymers with a high cationic charge density which function as transfection reagents based on their ability to compact DNA or RNA into complexes. This paper describes the application of lyophilized PEI/siRNA complexes based on a novel polyethylenimine. By fractionation of a commercially available 25-kDa PEI using gel permeation chromatography, a low molecular weight polyethylenimine (PEI F25-LMW) with superior transfection efficacy and low toxicity in various cell lines is obtained. Complexes formed in 5% glucose, but not in 150 mM NaCl, can be lyophilized and reconstituted without loss of transfection efficacy. Furthermore, PEI F25-LMW is able to complex and fully protect siRNAs against nucleolytic degradation, and delivers siRNAs into cells where they display bioactivity. Upon lyophilization and reconstitution of PEI F25-LMW-based siRNA complexes, siRNAs are still able to efficiently induce RNAi. To further demonstrate their applicability, lyophilized PEI/siRNA complexes are employed for targeting of the growth factor VEGF. Treatment of PC-3 prostate carcinoma cells with fresh or with lyophilized complexes results in decreased cell proliferation in different assays due to the siRNA-mediated downregulation of VEGF. In conclusion, siRNAs can be applied in lyophilized formulations, and lyophilized PEI F25-LMW-based siRNA complexes represent a powerful, inexpensive, non-toxic and simple ready-to-use platform for the specific and efficient targeting of genes in vitro.