Protein transduction domains (PTDs) have been used to deliver a variety of biologically active cargo across cellular membranes. However the potential of PTDs to mediate transport of nanoparticular structures into the cytoplasm bypassing the endosomal compartment remains unclear. Cell‐permeable virus‐like particles (VLPs) harboring a marker gene based on hepatitis B virus nucleocaspids were established. Cell permeability was achieved by fusion with translocation motif (TLM)‐PTD. Electron and confocal microscopy revealed that these VLPs translocate as complete particles across the plasma membrane and transverse the cytoplasm toward the nucleus. Inhibition of endocytosis did not affect translocation of these VLPs into the cytoplasm. Based on these particles, a gene transfer system was developed. To this end the particles were loaded with DNA‐encoding small hepatitis B virus surface antigen (SHBs) or green fluorescence protein (eGFP) that served as marker genes. Although the DNA‐packaging efficiency was very low, applying the appropriate number of VLPs to primary human hepatocytes a gene transfer efficiency of approximately 95% was observed. In conclusion, the TLM‐PTD has the potential to mediate efficient transfer of assembled particles and its cargo, nucleic acids, into primary human hepatocytes. This provides the basis for development of novel transducible therapeutic or diagnostic particles. (HEPATOLOGY 2005;42:1300–1309.)