Understanding cellular uptake and intracellular processing of nonviral gene delivery systems is a key aspect in developing more efficient vectors. In this study, the impact of clathrin- and caveolae/lipid-raft-dependent endocytosis on cell entry and overall transfection efficiency of polyethylenimine (PEI) polyplexes was evaluated. Most remarkably, the internalization pathway mediating successful transfection depended on both cell type and polyplex type applied. Colocalization studies with transferrin and cholera toxin B revealed that at least two specific endocytosis pathways--the clathrin-dependent and the lipid-raft-dependent--mediated cellular uptake of PEI polyplexes. With the help of specific uptake inhibitors (chlorpromazine and filipin III), cell-line-dependent variations regarding the route of successful transfection were observed (HUH-7, COS-7, HeLa). In COS-7 cells, the clathrin-dependent pathway was the main contributor to the transfection process. In HUH-7 cells, gene transfer by linear PEI polyplexes succeeded mainly via the clathrin-dependent route, whereas transfection by branched PEI polyplexes was mediated by both pathways. In HeLa cells, both pathways were able to mediate successful gene delivery. However, the lipid-raft-dependent pathway was more relevant. The study also revealed that the concentration window between specific inhibitory function and nonspecific toxicity of the uptake inhibitors was very narrow.