Arginine-rich cell-penetrating peptides (CPPs) including Tat, Penetratin and oligoarginine peptides are a series of short peptides that can be efficiently internalized into cells and have been widely used as carriers for intracellular delivery of bioactive molecules. In the early phase of the study, CPPs, as well as their conjugates, were thought to rapidly enter cells by direct penetration through membranes, which was later found to be an experimental artifact that was concluded from observations in fixed cells. Although re-evaluation using living unfixed cells revealed that endocytosis has a major role in internalization of these peptides, there are a number of studies reporting that, even if fixation is avoided, direct translocation across plasma membranes and cytosolic distribution of arginine-rich CPPs are still observed in cells without membrane perturbation. In addition, amphiphilic counteranions such as pyrenebutyrate dramatically accelerate direct translocation of these peptides into cells. These results suggest that there are at least two pathways, ie, endocytosis and direct translocation, both of which would contribute to cellular internalization of arginine-rich CPPs. In this review, we first introduce the story of fixation artifact, which indeed led to the critical progress in CPP study, and then summarize the current understanding for direct translocation of arginine-rich CPPs. Comprehensive understanding of direct translocation of these peptides and its mechanistic elucidation would provide useful knowledge for developing methodologies that would enable efficient intracellular delivery.