Adenoviruses are a promising vector system for future gene therapy of heart muscle diseases. The promiscuous tissue tropism of adenoviruses, however, may lead to the undesirable expression of putative therapeutic genes in nontarget cells and hence to considerable safety limitations for this vector system. To restrict gene expression to cardiomyocytes we constructed an adenoviral vector (Ad-mlcLuc) in which the luciferase gene is under the control of the ventricle-specific myosin light chain-2 (mlc-2v) promoter. For controls, we constructed a recombinant adenovirus without promoter (Ad-Luc) and one with the Rous sarcoma virus (RSV) promoter (Ad-rsvLuc). Our data demonstrate that the newly established viral vector Ad-mlcLuc was specifically active in rat neonatal cardiomyocytes in vitro but not in three established cell lines. Injections of the recombinant adenoviruses into the cardiac cavity of neonatal rats resulted in myocardial specific gene expression of Ad-mlcLuc in vivo, despite the fact that viral DNA was detected by PCR at different levels in all tissues investigated. In vitro and in vivo, Ad-mlcLuc was exclusively active in cardiac muscle cells, reaching 8-9% of the RSV-induced luciferase activity. Direct injection of Ad-mlcLuc into thigh muscle gave only background luciferase activity (0.05% of Ad-rsvLuc). Therefore, in the adenoviral system, the mlc-2v promoter allows heart-specific expression of a foreign gene thus providing a promising tool for gene transfer targeted to the myocardium.