Glycosaminoglycans (GAGs) are negatively charged polysaccharides that are found, e.g. on cell surface. GAGs have been reported to influence gene transfection. We have previously reported that cationic lipid-mediated gene transfection can be improved by combining a small polyethylenimine (PEI) with cationic lipids. In the present study, we examined if GAGs have any effect on the synergism of small PEIs and other cationic carriers. We used wild-type CHO (GAG+) and pgsB-618 cells (GAG-). Transfection efficiency was studied using lacZ and GFP reporter genes. We found that GAGs decreased the overall level of transgene expression in a reagent-dependent manner, but the synergism caused by low-MW PEIs was less affected. There were no major differences between cell lines in cellular uptake or intracellular localization of plasmids when measured with flow cytometry and confocal microscopy, respectively. In conclusion, cell-surface GAGs interfere with transfection efficiency of different cationic reagents, but that is not necessarily related to the synergy of small PEIs and cationic lipids.