Efficient transfer of proteins or nucleic acids across cellular membranes is a major problem in cell biology. Recently the existence of a fusogenic sequence was predicted in the junction area of the PreS2- and S-domain of the hepatitis-B virus surface antigens. We have identified cell permeability as a novel property of the PreS2-domain. Cell permeability of PreS2 is not restricted to hepatocytes. PreS2 translocates in an energy-independent manner into cells and is evenly distributed over the cytosol. Detailed analysis revealed that cell-permeability is mediated by an amphipatic alpha-helix between amino acids 41 and 52 of PreS2. Destruction of this translocation motif (PreS2-TLM) abolishes cell permeability. PreS2-TLM per se can act as a shuttle for peptides and functional proteins (such as EGFP). This permits the highly specific modulation of intracellular signal transduction by transfer of peptides competing protein-protein interactions as demonstrated by specific inhibition of TNFalpha-dependent activation of c-Raf-1 kinase. Moreover in vivo functionality was demonstrated by PreS2-TLM-dependent protein transfer into primary bone marrow cells and into the liver. The amphipatic motif is conserved between the different hepatitis-B virus subtypes, and the surface proteins of avian and rodent hepadnaviruses exhibit similar amphipatic peptide sequences. In respect to hepatitis-B virus-infection, the PreS2-TLM could represent the postulated fusion peptide and play a crucial role in the internalization of the viral particle.