Top of pageAbstract One of the most promising non-viral vectors is the cationic polymer polyethylenimine (PEI), a well-known cell culture transfection reagent. In particular, the latest generation of linear PEI is more efficient than the first generation of branched polymers. Various delivery routes of small size defined PEI polyplexes formulated in a 5% glucose solution can be used (intravenous, intracerebral, intraperitoneal, intracardiac, instillation |[hellip]|). Intravenous injection (IV) is the route of choice in many gene therapy applications. Among the different routes of IV injection tested (caudal, jugular and retro-orbital), the retro-orbital route appears to be the simplest and the most efficient one to deliver the gene of interest in the organs studied (i.e. lung, liver, spleen, heart and kidney). The purpose of our work was to compare different parameters of DNA injection complexed with L-PEI injected by retro-orbital route. Different amount of pCMVLuc plasmid was injected complexed with in vivo-jetPEI|[trade]| in 22|[ndash]|24 grams OF1 female mice. Among the DNA quantity injected (10 |[mu]|g to 70 |[mu]|g) at N/P ratio of 8, we have shown that the quantity of 40 or 50 |[mu]|g gives the most efficient plasmid expression. For this amount, the best result was obtained by injecting 400 or 600 |[mu]|l instead of 200 |[mu]|l. As the efficiency and the cytotoxicity depend on the molecular weight of the polymer, we compared different polymer length and showed that the commercial in vivo-jetPEI|[trade]| by being noncytotoxic is the most efficient in gene delivery. The pharmacokinetic and organ distribution profile was also determined with 40 |[mu]|g of plasmid between 6 and 72 hours after the injection of the plasmid. Taken together, our data showed that the IV retro-orbital injection is the route of choice to inject plasmid DNA complexed with linear polyethylenimine. Moreover, we have also explored the promising potential of in vivo-jetPEI|[trade]| to deliver active siRNAs injected by retro-orbital route and shown that in vivo-jetPEI|[trade]| can deliver siRNAs silencing a co-transfected target gene expressed in lungs after intravenous injection in mice.