Cationic lipids are widely used as vectors to deliver DNA into mammalian cells in vitro and in vivo. However, cationic lipid/DNA lipoplexes induce an inflammatory response, characterized by pro-inflammatory cytokine secretion, which severely limits their use. The main goal of this work is to identify the organs and the cell type involved in TNF-alpha secretion after lipoplex injection. We determined the kinetics of distribution of the cationic lipid/DNA complex in blood, lung, liver and spleen and quantified the TNF-alpha amount in organ homogenates and in the serum at different points of times. Increase in TNF-alpha production was only observed in the spleen and no significant increase of TNF-alpha production could be observed in the other organs. Fractionation of spleen cells revealed that macrophages were mainly responsible for TNF-alpha secretion. This observation was verified in vivo by using macrophage-removing agents. In conclusion, we show here that the TNF-alpha secreted in the serum after intravenous injection of lipoplexes comes mainly from the splenic macrophages.