The retinal pigment epithelium (RPE) is a potential tissue for gene therapy. We recently demonstrated that pegylation of lipoplexes prevents their aggregation in the vitreous of the eye. However, pegylation of lipoplexes may affect their gene transfer capacity. Therefore we studied the effect of pegylation of lipoplexes on the transfection of RPE cells. "Pre-pegylated" lipoplexes were prepared by mixing pDNA with pegylated liposomes, while "post-pegylated" lipoplexes were obtained by pegylation of (non-pegylated) cationic liposome/DNA complexes with PEG-ceramides. Pre-pegylation of lipoplexes severely inhibited their transfection efficacy. The poor transfection was attributed to an inefficient and slower internalization of pre-pegylated lipoplexes by RPE cells, compared to non-pegylated lipoplexes. In addition, pre-pegylated lipoplexes also remained entrapped in the endosomes of the RPE cells. In contrast, post-pegylation of the lipoplexes with PEG-ceramides strongly improved their transfection efficiency. As PEG-ceramides are believed to leave the lipoplexes upon contact with the cell membranes, this "de-pegylation" results in non-pegylated lipoplexes which successfully escape from the endosomes. In conclusion, post-pegylation of lipoplexes with PEG-ceramides appears to be an attractive strategy to deliver therapeutic DNA to RPE cells as (a) it prevents the lipoplexes from aggregation in vitreous and (b) de-pegylation upon contact with RPE membranes results in successful DNA delivery.