Lipopolythioureas (LPT) are original non cationic systems representing an alternative to cationic lipids. Their high transfection efficiency prompted us to investigate further their biophysical properties, and in particular how thiourea lipids interact with DNA. The interaction of lipopolythiourea with DNA was investigated by fluorescence correlation microscopy (FCS). Influence of the lipid length and nature of the thiourea head on the thiourea/DNA interaction were studied. FCS revealed a strong interaction between lipopolythiourea and DNA, occurring at 1 equivalent of a thiourea lipid by a DNA phosphate group, and leading to a condensed plasmid state. From previous in vitro experiments, we could conclude that the lipid leading to the more condensed state of DNA was also the more efficient to transfect cells.