Arginine-rich cell-penetrating peptides (CPPs), including oligoarginine peptides, have been widely used as a tool for intracellular delivery of various molecules with low membrane permeability. We previously reported the enhanced cytosolic entry of arginine-rich CPPs by the attachment of a short peptide segment, the penetration accelerating sequence (Pas). In this study, the importance of hydrophobic sequences, especially phenylalanine residues, in the Pas segment was demonstrated for this enhanced translocation through cell membranes. The advantage of using Pas for intracellular delivery was particularly marked for delivering cargoes with a relatively small molecular weight, such as bioactive peptides. In addition, the results of this study indicate the important roles that the total hydrophobicity of the PasR8 conjugates play in cytosolic translocation and the eventual bioactivity thus attained.