Structural dynamics of the polyethylenimine DNA and poly(L-lysine) DNA complexes (polyplexes) was studied by steady-state and time-resolved fluorescence spectroscopy using the fluorescence resonance energy transfer (FRET) technique. During the formation of the DNA polyplexes, the negative phosphate groups (P) of DNA are bound by the positive amine groups (N) of the polymer. At N/P ratio 2, nearly all of the DNA's P groups are bound by the polymer N groups: these complexes form the core of the polyplexes. The excess polymer, added to this system to increase the N/P ratio to the values giving efficient gene delivery, forms a positively charged shell around the core polyplex. We investigated whether the exchange between the core and shell regions of PEI and PLL polyplexes takes place. Our results demonstrated a clear difference between the two studied polymers. Shell PEI can replace PEIs previously attached to DNA in the polyplex core, while PLL cannot. Such a dynamic structure of PEI polyplexes compared to a more static one found for PLL polyplexes partially explains the observed difference in the DNA transfection efficiency of these polyplexes. Moreover, the time-resolved fluorescence spectroscopy revealed additional details on the structure of PLL polyplexes: in between the core and shell, there is an intermediate layer where both core and shell PLLs or their parts overlap.