The aim of the present study was to compare different regularly used transfection agents, i.e. poly(L-lysine) (PLL), branched poly(ethyleneimine) (BPEI), linear PEI (LPEI), poly(2-(dimethylamino)ethyl methacrylate) (pDMAEMA) and 1,2-dioleyl-3-trimethylammonium-propane (DOTAP), regarding their in vivo transfection behavior after intravenous administration. An attempt was made to study whether the in vitro behavior of these transfectants is relevant for the in vivo situation in terms of their transfection efficiency and serum aggregation properties. In vivo, at an N/P ratio of 5, transfection mediated by the various cationic transfectants mainly occurred in the lungs. The order of decreasing lung transfection was: DOTAP > LPEI > pDMAEMA > BPEI > PLL. Similar rankings were found in in vitro experiments regarding the extent of serum-induced aggregation, suggesting that in vitro studies are certainly relevant for the in vivo situation. As a whole, the in vitro data suggest that the induction of aggregates in the circulation is a major mechanistic factor underlying the phenomenon of dominant lung transfection.