The impact of the length of gemini surfactant spacer on complexation and condensation of calf thymus DNA by cationic mixed phospholipid/gemini liposomes was investigated by monitoring the conformational changes of DNA by circular dichroism and the lipid hydration level by the emission characteristics of the fluorescent probe laurdan included in the lipid bilayer. The length of the spacer was shown to influence, on one hand, the hydration level and the organization of the corresponding liposomes and, on the other, the variation of lipid hydration level and the DNA conformation upon complexation. In fact, in correspondence with the longest spacer we observed more hydrated liposomes, probably organized in domains, a higher extent of dehydration promoted by the addition of DNA, and a minor extent of DNA conformational change. The physicochemical features of lipoplexes were shown to depend on the [cationic headgroup]/[DNA single base] ratio.