The mechanism in which small cationic oligopeptides are able to reach the cytosol of cells is controversial. Macropinocytosis has been recently suggested as a major mechanism for internalization of these peptides. In this report, the involvement of macropinocytosis on cytosolic localization of oligoarginine was quantitatively investigated in HeLa cells. Using a method which allows for the separate measurement of cytosolic versus vesicular oligopeptide, the results show that neither macropinosome nor filopodia formation correlates with cytosolic delivery of oligoarginine. Additionally, unlike macropinocytosis, the cytosolic delivery of oligoarginine was not inhibited by incubation at 16 degrees C, or by treatment with amiloride. Oligoarginine treatment does not contribute to leakage from endocytic vesicles, indicating the lack of endosomolytic properties. Finally, the amount of oligoarginine found in the cytosol was not substantially increased after coincubation with EGF, a known stimulator of macropinocytosis. Taken together, these data indicate that membrane transduction of oligoarginine occurs separately from macropinocytosis in HeLa cells.