One of the major issues in myocardial gene therapy is poor transfection efficiency. The herpes simplex virus protein VP22 is known to facilitate intercellular protein transport. Not only VP22 but also VP22-linked protein are exported from the cytoplasm of cells, in which it is synthesised endogenously, and transferred to surrounding cells, where it is translocated into the nuclei. However, the feasibility and efficiency of the intercellular trafficking properties of VP22-linked protein in the myocardium has not been clarified. Rat hearts were transfected by direct intramyocardial injection of naked plasmid vectors encoding either lacZ or VP22-linked lacZ. At day 5 following transfection, similar numbers of cardiomyocytes surrounding the injection sites showed beta-galactosidase (beta-gal) expression in the cytoplasm in both groups. In addition to this, following transfection of VP22-linked lacZ, most of the cardiomyocytes adjacent to the cytoplasmic-positive cells demonstrated nuclear-localised beta-gal expression. The number of these nuclear-positive cardiomyocytes, which are thought to be secondary protein-transported cells, was 4.3-fold greater than that of primary transfected, cytoplasmic-positive cells. Western blot analysis demonstrated that the amount of targeted protein expression is 2.9-fold greater following VP22-lacZ transfection (VP22-linked beta-gal; approximately 40 kDa bigger than wild-type beta-gal) compared with lacZ transfection (wild-type beta-gal). This data highlights the efficiency of the VP22-mediated intercellular protein delivery in the myocardium following in vivo gene transfection and suggests that the VP22-mediated effect is useful in enhancing the efficacy of myocardial gene therapy.