The capacity of VP22 chimeric proteins to spread from the primary transduced cell to surrounding cells could improve gene therapy approaches, especially in cancer therapy. However, there are conflicting data about VP22-mediated intercellular trafficking in different studies. To assess the role of VP22 in gene therapy of hepatocellular carcinomas (HCCs) we constructed expression vectors for N- and C-terminal versions of VP22-p53 fusion proteins and investigated the VP22-mediated shuttle effect in hepatoma cells by cotransfection experiments. VP22-mediated trafficking was not detectable in hepatoma cells in vitro by fluorescence microscopy, but reporter gene transactivation assays demonstrated intercellular trafficking of functional VP22-p53 in vitro. For in vivo experiments, the recombinant adenoviruses Ad5CMVp53 and Ad5CMVp53-VP22 were constructed. In contrast to the in vitro experiments intercellular trafficking of VP22-p53 could be observed in subcutaneous tumors of hepatoma cells by fluorescence microscopy, indicating a stronger shuttle effect in solid tumors compared to cell culture experiments. Because spread of p53-VP22 in liver tumors was correlated with enhanced apoptosis of hepatoma cells VP22-mediated trafficking of potential therapeutic proteins may improve the results of gene therapy of HCCs.