The cell membrane is a biological boundary that separates the interior of cellsfrom the extracellular environments. While small non-polar molecules readilycross cell membranes, the cell membrane precludes the passage of mostmacromolecules, including proteins, DNAs, and RNAs. However, basiccell-penetrating peptides (CPPs) can enter cells, and can co-transport an array ofnormally impermeable molecules into cells. In this report, we identified factorsthat determine the mechanisms of CPPs entry. Three arginine-rich CPPs (R9,SR9,and HR9) were studied in live human, plant, and bacterial cells.Pharmacological and physical treatments were used to elucidate the nature of thetransport mechanism. The route of internalization was relatively unaffected bycell type, but was dependent on the nature of the CPP as well as the nature of thetransported cargo.