Despite the widespread interest in peptide carriers, the mechanisms underlying the cellular translocation of CPPs are poorly understood. Early work relied upon fluorescence imaging or flow cytometry analysis of chemically fixed cells to examine intracellular localization of fluorescently labeled peptides in the absence or presence of cargo. According to these experiments peptides appeared to be internalized very rapidly within minutes even at 4 °C. From such observations it was concluded that CPPs penetrate cell membranes by an energy-independent mechanism [46] [47] [48] [49] [50] . Although it had been reported quite early on that certain fixation procedures may cause artefacts leading to an overestimation of the cellular uptake rates [51] [52] [53] the dimension of this problem was not commonly recognized until a side by side comparison of fixed and living cells was published [54] . Based on these findings, many groups re-examined their data. However, despite considerable technical improvements, there are still puzzling controversial results concerning the exact mechanism of CPP uptake. Though in most cases endocytosis has been suggested to be the main route of internalization (Fig.  (1A) ), substantial difficulties are encountered identifying the exact pathway ( [42, 55] and references therein). Prior to endocytosis CPPs interact electrostatically with the extracellular matrix of the cell surface mostly through binding to negatively charged glycosaminoglycans, i.e. heparan sulfate proteoglycans [56] [57] [58] [59] . Recent studies indicate that the uptake mechanism of CPPs can be influenced by the attachment of cargos. For example, Richard et al. [54, 60] reported a colocalization of Tat 48-59 with markers of clathrin-mediated endocytosis, whereas Fittipaldi et al. [61] found a caveolae/lipid raft-dependent process for a Tat-GFP fusion protein and Wadia et al. [62] described a macropinocytotic uptake pathway for a fusion construct of Tat peptide with Cre recombinase. In summary, the precise mechanism of internalization remains elusive and strongly depends on the properties of both CPP and cargo as well as on the transfection conditions and the cell lines used [63] [64] [65] [66] [67] [68] .