Many cell-penetrating peptides (CPPs) bind to glycosaminoglycans (GAG) located on the extracellular side of biological tissues. CPP binding to the cell surface is intimately associated with clustering of surface molecules and is usually followed by uptake into the cell interior. We have investigated the uptake mechanism by comparing CPPs which bind, but cannot induce, GAG clustering with those which do induce GAG clustering. We have synthesized the tryptophan-labeled CPP nona-l-arginine (WR(9)) and its monodispersely PEGylated derivate (PEG(27)-WR(9)) and have compared them with respect to glycan binding, glycan clustering, and their uptake into living cells. Both CPPs bind to the GAG heparin with high affinity (K(D) ∼ 100 nM), but the PEGylation prevents the GAG clustering. Thus, it is possible to uncouple and analyze the contributions of GAG binding and GAG clustering to the biological CPP uptake. The uptake of PEG-WR(9) into CH-K1 cells is confined to intracellular vesicles, where colocalization with transferrin attests to an endocytic uptake. Transfection experiments with plasmid DNA for GFP revealed poor GFP expression, suggesting that endocytic uptake of PEG-WR(9) is compromised by insufficient release from endocytic vesicles. In contrast, WR(9) shows two uptake routes. At low concentration (<5 μM), WR(9) uptake occurs mainly through endocytosis. At higher concentration, WR(9) uptake is greatly enhanced, showing a diffuse spreading over the entire cytoplasm and nucleus-a phenomenon termed "transduction". Transduction of WR(9) leads to a higher GFP expression as compared to PEG-WR(9) endocytosis but also damages the plasma membrane as evidenced by SYTOX Green staining. The results suggest that GAG binding without and with GAG clustering induce two different pathways of CPP uptake.