In search of optimizing siRNA delivery systems for systemic application, one critical parameter remains their stability in blood circulation. In this study, we have traced pharmacokinetics and biodistribution of each component of siRNA polyplexes formed with polyethylenimine 25 kDa (PEI) or PEGylated PEIs by in vivo real-time gamma camera recording, SPECT imaging, and scintillation counting of blood samples and dissected organs. In vivo behavior of siRNA and polymers were compared and interpreted in the context of in vivo stability of the polyplexes which had been measured by fluorescence fluctuation spectroscopy (FFS). Both pharmacokinetics and biodistribution of polymer-complexed siRNA were dominated by the polymer. PEGylated polymers and their siRNA polyplexes showed significantly less uptake into liver (13.6-19.7% ID of PEGylated polymer and 9.5-10.2% ID of siRNA) and spleen compared to PEI 25 kDa (liver deposition: 36.2% ID of polymer and 14.6% ID of siRNA). With non-invasive imaging methods we were able to predict both kinetics and deposition in living animals allowing the investigation of organ distribution in real time and at different time points. FFS measurements proved stability of the applied polyplexes under in vivo conditions which explained the different behavior of complexed from free siRNA. Despite their stability in circulation, we observed that polyplexes dissociated upon liver passage. Therefore, siRNA/(PEG-)PEI delivery systems are not suitable for systemic administration, but instead may be useful when the first-pass effect is circumvented, which is the case in local application.