The intercellular trafficking property of the herpes simplex virus type 1 tegument protein VP22 makes it a promising tool for overcoming low transduction efficiencies in gene therapy. However, recent reports suggest not only that VP22 cannot facilitate intercellular spreading and that trafficking of VP22 fusion proteins results from artifacts of cell fixation only. To provide direct evidence for the presence or absence of VP22-mediated intercellular trafficking, we generated an adenoviral vector with a dual expression cassette for VP22 fused to green fluorescent protein (VP22 GFP) and DsRed under the control of distinct human cytomegalovirus immediate-early enhancer/promoter regions. Using this vector, we were able to distinguish clearly between primary transduced cells and cells taking up VP22GFP by intercellular trafficking. To our knowledge, for the first time, we could demonstrate by live-cell confocal fluorescence microscopy that VP22GFP can be found intracellularly in unfixed recipient cells. The extent of VP22 spread was similar in paraformaldehyde-fixed cells and unfixed cells as demonstrated by fluorescence-activated cell sorting analysis. We thus confirmed the ability of VP22-mediated intercellular trafficking in live unfixed cells.