Naturally occurring and synthetic short arginine containing protein transduction domains (PTDs), including HIV1 TAT, poly-Arg and Antp, have been used to deliver a wide variety of macromolecular, biologically active therapeutic cargo into cells, including peptides, proteins, antisense oligonucleotides and liposomes, in vitro and to treat pre-clinical models of cancer and stroke. PTDs enter cells in a rapid, receptor-independent fashion. Recently, large TAT-fusion proteins (in excess of 30,000 Da) were shown to transduce into cells by fluid-phase macropinocytosis, a specialized form of endocytosis that is independent of caveolae, clathrin and dynamin. However, it remains controversial as to whether or not PTD peptides (1000-5000 Da) enter cells via macropinocytosis and/or through an unknown alternative mechanism. Due to strong ionic interactions with the cell surface, previous measurements of PTD peptide internalization were inaccurate. Cationic PTD peptides containing variable numbers of arginine residues and conditions entered cells exclusively through macropinocytosis. In addition, no PTD peptide was found to enter cells at 4 degrees C, a long held assumption of transduction. Taken together, these observations provide a solid scientific basis for the development of novel biologically active transducible anticancer PTD peptide therapeutics.