The phosphorothioate(PS)-stimulated cellular uptake of naked short interfering RNA (siRNA) into mammalian cells indicates a promising new mechanistic strategy because it makes use of a caveosomal, rather than an endosomal pathway, which is used by the majority of known delivery systems. This PS-stimulated mode delivers large amounts of siRNA primarily into the perinuclear space which is related to measurable though moderate target suppression. The observed limited efficacy seems to be related to intracellular trapping of siRNA. Here, we studied the intracellular localisation of siRNA and Argonaute 2 (Ago2), the major component of the RNA interference (RNAi) machinery, by density gradient centrifugation and fluorescence microscopy after PS-stimulated delivery or transfection with Lipofectamine 2000. The two cell lines ECV-304 and SKRC-35 both take up siRNA in the PSstimulated mode but only ECV-304 shows RNAi, i.e. siRNA-mediated suppression of lamin A/C expression, whereas SKRC-35 does not. This lack of RNAi in the latter cell line seems to be due to a block of an intracellular siRNA translocation process. This study provides strong evidence for the view that co-localisation of siRNA and Ago2 in the vicinity of the rough endoplasmic reticulum (rER) in ECV-304 cells is related to target inhibition, whereas density gradient fractionation of cell organelles shows a lack of co-localisation in SKRC-35 cells in which RNAi does not occur after the PS-mediated delivery. In summary, we propose to exploit this dual cell system to identify important steps of intracellular trafficking of siRNA after PS-mediated delivery that are crucial for its biological activity and which seem to be of general importance for the understanding of the intracellular trafficking and release of siRNA.