Cationic amphipathic histidine rich peptides possess high plasmid DNA and siRNA delivery capabilities. To further understand the pH responsive siRNA delivery process and evaluate the capabilities of such peptides we have investigated their ability to mediate specific silencing of endogenous GAPDH gene activity in MCF-7 and A549 cells and compared this with plasmid DNA delivery. A substantial and selective reduction of both GAPDH activity and expression was achieved using pH responsive peptide vectors, which compared favourably with that mediated by commercially available non-viral vectors in terms of efficacy and toxicity. Furthermore, by comparing the efficacy of both gene delivery and silencing mediated by a series of such peptides, their sensitivities to known inhibitors of endocytotic processes, and their route of uptake via confocal live cell imaging, we show that both plasmid DNA and siRNA are internalised via endocytosis. However siRNA entry facilitated by LAH4-L1, proceeds via a cholesterol dependent mechanism, in contrast to DNA transfer which is associated with clathrin dependent endocytosis. Furthermore, using peptides that respond at increasingly acidic pH, we demonstrate that the route of entry for the siRNA that ultimately mediates silencing is peptide specific and whilst some pH responsive peptides promote the escape of labelled siRNA from endosomes, others may promote entry via alternative mechanisms. (C) 2011 Elsevier B.V. All rights reserved.