Pep-1 is a cell penetrating peptide (CPP) derived from the nuclear localization sequence of Simian Virus 40 large antigen T and from reverse transcriptase of Human Immunodeficiency Virus. Although it has been successfully used to transport proteins into cells, its action at the molecular level is not yet clear, mainly the local environmental factors that condition partition and translocation. Characterization in aqueous medium and quantification of partition into bilayers were carried out. Dynamic light scattering studies show that pep-1 self-associates in aqueous medium. The role of the bilayer phase, anionic lipids, ionic strength of the medium, reducing agents and pep-1 concentration on the extent and kinetics of partition were studied. Unlike others cationic CPP (e.g. penetratin) pep-1 has a high affinity to neutral vesicles (Kp = 2.8 x 10(3)), which is enhanced by anionic lipids. In a reduction environment partition is strongly inhibited (Kp = 2.2 x 10(2)), which might be a key-feature in the biological action of pep-1. Peptide incorporation takes place in the millisecond time-range to the lipidic interfaces. These environmental factors are systematized to enlighten how they help cellular uptake.