The ability to translocate the cellular membranes and gain access to the cell interior, including the different cellular compartments, still remains a major obstacle in current drug development. Peptide mediated delivery of bioactive molecules appears to be a technology that in many aspects is superior to commonly used delivery agents. Reported high delivery yield, low toxicity and the possibility to add diverse modifications to the peptide backbone make peptides an excellent candidate for future drug delivery platforms. So-called cell-penetrating peptides (CPPs), also often referred to as protein transduction domains (PTDs), Trojan peptides or membrane translocating sequences (MTS), have in recent years shown great potential in the field of drug delivery. Current publications show that CPPs can deliver a wide range of bioactive molecules such as proteins, peptides, oligonucleotides (ON), and nano-particles to a variety cell types and to different cellular compartments, both in vivo and in vitro. The peptides named CPPs vary greatly in size, amino acid sequence, and charge, but share the common feature that they have the ability to rapidly translocate the plasma membrane and enable delivery to the cytoplasm or nucleus [1]. The idea to use peptides used as delivery vectors, i.e. CPPs, originates from so-called membrane shuttling proteins such as the Drosophila homeobox protein Antennapedia, the HIV-1 transcriptional factor TAT, and the capsid protein VP22 from HSV-1. The field started in 1988, when Green et al. showed that the viral protein TAT rapidly translocate over cellular membrane, into the cytoplasm [2]. Later, the same properties were shown for a Drosophila homeobox protein. In 1994, Alain Prochiantz' group demonstrated that a short, 16 amino acid (aa) peptide derived from the third loop of the Antennapedia protein was responsible for the cellular translocation of the whole protein [3]. This pioneering work initiated the whole field using peptides as efficient delivery vectors for bioactive compounds, cell-penetrating peptides. Since then, a myriad of peptides has been reported to have cell-penetrating properties. The peptides originate from different classes; either naturally occurring peptide sequences such as virally derived (TAT, VP22), from transcription factors (pAntp), chimeric peptides (transportan, MGP) or synthetic (poly-arginines, Pep-1), cf. Table 1. However, due to difficulties in understanding the true mechanisms of CPP cellular uptake, the classification of CPPs still remains to be clarified. Although great achievements in studies of CPP have been attained, a clear description of their properties is still not defined. The CPP field has been under constant change during the years and the uptake mechanism still remains ambiguous. Several attempts have been made in order to elucidate the true mechanism of peptide mediated uptake, but the results are divergent between different reports and experiments. Even when using the same peptide, results vary between different publications. Furthermore, it seems apparent that different peptides utilize different uptake pathways [4]. Early studies on CPP translocation mechanisms suggested that the internalization of these peptides was not inhibited by depletion of the cellular adenosine triphosphate (ATP) pool, low temperature (+4 °C), or by inhibitors of endocytosis [5]. Neither did chemical modifications of the peptide sequences, such as the synthesis of retro-, enantio- or retroenantio-analogs, appear to affect the internalization properties [6]. Therefore, translocation was thought to result from direct transfer through the lipid bilayer of the cell membrane. Formation of inverted micelles was a proposed mechanism for these uptakes, where cationic residues interact with the negatively charged plasma membrane followed by invagination of tryptophans into the membrane, inducing inverted micelle formation [6]. The postulated mechanism of inverted micelle formation seems to explain some aspects of CPP translocation and still appear to hold for some peptides used in cellular delivery of bioactive molecules. Later studies, on the other hand, show that the CPP translocation mostly is an energy-dependent process [7]. Proposed mechanisms involve extracellular heparane sulfate [8] and different types of endocytosis [9,10] such as macropinocytosis, clathrin-dependent-, caveoale-dependent- or clathrin- and caveoale-independent endocytosis. It seems to us that these processes do not necessarily contradict each other rather demonstrating that peptide mediated membrane translocation is mediated by several different pathways simultaneously, or that different peptides utilize diverse uptake mechanisms depending on their cargo and biophysical properties [11]. The main explanation today seems to be that endosomal pathways contribute to the major route of uptake of CPPs, although several reports show an uptake independent of endocytosis [4,12,13]. The uptake mechanism could also be altered by different cargoes, if the peptides form a stable complex with its cargo, if the cargo is covalently bound to a CPP or how the cargo is attach [14] (Fig. 1).