Previous studies using different techniques have shown that adenoviral-mediated gene transfer to different tissues, including the kidney, is more efficient in neonatal mice. In this study, we report a simple technique that allows an efficient and long term expression of β-galactosidase (β-gal) in the heart of newborn mice. Newborn and adult C57BL6/J mice were subjected to a single retro-orbital venous plexus injection of recombinant adenoviral vectors (rAd) (2 x 10 particles/ g body weight) carrying the lac Z gene. Seven days after the injection, positive β-gal staining was systematically observed in the heart, lung, intestine, liver, kidney and spleen of newborn mice. However, only the heart showed persistent expression of β-gal one year after the initial injection. In contrast, adult mice showed only significant but transient β- gal expression mainly in the liver. In summary, we have found that a single retro-orbital intravenous injection can be used to establish a long-term adenoviral-mediated gene transfer to cardiac cells of newborn mice.