Endocytosis of non-viral vectors delivers them first to early endosomal compartments followed quickly by late endosomes. Vectors are then subject to possible sequestration to lysosomes. The acidic environment within lysosomes activates acidic nucleases such as Deoxyribonuclease II (DNase II) which cause vector degradation. Previous research has identified DNase II as a lysosomal barrier to transfection; however, the significance of this barrier to cationic gene transfer in nondividing endothelium has yet to be determined. To probe the contribution of individual proteins to the non-viral gene transfer mechanism, we introduce the use of RNA interference (RNAi). RNAi can be used to target specific transcripts for degradation and knock down the corresponding protein. Here, we probe the significance of DNase II knock down in the transfer of nucleic acid to nondividing human aortic endothelial cells (HAECs) in vitro. We transfected HAECs at or near confluence with short-hairpin RNA (shRNA) and small interfering RNA (siRNA) targeted against human DNase II using Lipofectamine 2000. Several shRNA and siRNA sequences targeting DNase II were transfected along with inactive controls and DNase II knock down was verified using an acidic nuclease digestion gel assay. Cell lysates were incubated with lambda-phage DNA at pH 4.5 and run on an agarose gel to determine the relative nuclease activity. Comparing the treatment of different shRNAs and siRNAs revealed one shRNA sequence and two siRNA sequences which achieved a significant knock down. To determine the effect of DNase II knockdown on non-viral gene transfer, we transfected the shRNA or siRNA followed by a green fluorescent protein vector (pEGFP-N₃) using Lipofectamine 2000. DNase II knock down was verified in the presence of the plasmid transfection using the nuclease gel assay and cells were harvested for flow cytometry. To determine the transfection efficiency, raw flow cytometric data was analyzed using a numerical compensation technique to compute the GFP contribution compared with the negative control. The shRNA-mediated knock down of DNase II significantly decreases the transfection efficiency compared with untransfected controls, while the siRNA-mediated knock down of DNase II results in transfection efficiencies comparable to the negative and untransfected controls. Additionally, the shRNA knock down provokes cytotoxicity and inflammatory responses as measured by IL-8 ELISA. The cytotoxic effects are mitigated following siRNA transfection, which suggests that any deleterious effects are sequence dependent. The poor transfection efficiency of the GFP plasmid following the shRNA-mediated knock down of DNase II is most likely the result of the sequence dependent inflammatory and cytotoxic responses. The siRNA-mediated knock downs of DNase II result in no significant response and no change in the transfection efficiency. These results indicate that DNase II knock down does not improve cationic lipid transfection efficiency in nondividing human endothelium. Therefore, in the context of this experiment, we conclude that DNase II is not a rate limiting step to gene transfer. Furthermore, we have established that RNA interference can be used to probe the mechanism of gene transfer in vitro.