Recently, we have shown that polymethacrylates containing imidazole side groups (HYMIMMA) or acid functions (MA), which have similar buffering properties as polyethyleneimine, were not able to transfect Cos-1 cells, whereas polymers containing only tertiary amines (DMAEMA) do transfect Cos-1 cells (Dubruel, P. et al. Eur. J. Pharm. Sci. 2003, 18 (3-4), 211-220). In the present work, we investigated to what extent the differences in transfection activity are related to differences in cellular internalization and/or subcellular localization. Therefore, we synthesized a series of polymethacrylates containing primary amine functions, used for the coupling of the fluorescent Oregon Green probe. The polymers containing acid functions were labeled with an amine containing fluorescein derivative (5-aminomethyl)fluorescein hydrochloride. It is demonstrated that the endosomal release of the MA and HYMIMMA-based complexes might be the limiting step in the gene transfer process in Cos-1 cells.