To transfect cells, cationic polymers as well as cationic liposomes are widely investigated as carriers for both oligonucleotides and plasmid DNA. A major step in the successful intracellular delivery of the DNA is the release from its carrier. In this study, dual color fluorescence fluctuation spectroscopy (dual color FFS) was explored in order to characterize the intracellular dissociation of cationic polymer/oligonucleotide complexes. As a model, rhodamine green-labeled oligonucleotides (RhGr-ONs) were complexed with Cy5-labeled polymers of either high molar mass (Cy5-graft-pDMAEMA, 1700 kDa) or low molar mass [Cy5-poly(l-lysine), Cy5-pLL, 30 kDa]. The FFS results were compared with confocal laser scanning microscopy (CLSM) observations. CLSM proved that Cy5-graft-pDMAEMA/RhGr-ON complexes endocytosed by Vero cells dissociate in the cytoplasm: the polymer was only detected in the cytoplasm whereas the (released) RhGr-ONs accumulated in the nucleus. Transfecting Vero cells with Cy5-pLL/RhGr-ON complexes resulted, however, in colocalization of polymer and oligonucleotides in the nucleus. In the latter case, CLSM was not able to prove whether intact Cy5-pLL/RhGr-ON complexes were present in the nucleus or whether both components were located together in the nucleus without being associated. Dual color FFS, which monitors the movement of (dual labeled) fluorescent molecules, was able to answer this question. As a Cy5-pLL/RhGr-ON complex is multimolecular, i.e., it consists of many RhGr-ONs associated with many Cy5-pLL chains, it is both highly green and red fluorescent. Consequently, when Cy5-pLL/RhGr-ON complexes move through the excitation volume, the (green and red) detectors of the FFS instrument detect simultaneously a strong green and red fluorescence peak. Upon transfecting the Vero cells with Cy5-pLL/RhGr-ON complexes, FFS was indeed able to detect simultaneously green and red fluorescence peaks in the cytoplasm but never in the nucleus. From these results we conclude that the Cy5-pLL and RhGr-ONs present in the nucleus after transfection were not associated.