Adenoviruses are very attractive vectors for gene transfer into the cardiac muscle; however, their promiscuous tissue tropism, leading to an ectopic expression of the transgene, is a considerable practical limitation. To restrict expression of a reporter gene in cultured cardiomyocytes and in the heart of the rat, we have constructed a recombinant adenovirus (Ad-MLC2 beta gal) containing the beta-galactosidase gene under the control of the rat ventricle-specific cardiac myosin light chain 2 (MLC-2v) promoter. We show in this work that the MLC-2v promoter inside the adenoviral genome retains its cardiac specificity in vitro in cultured cardiomyocytes as well as in vivo in the animal heart. Northern blot studies after Ad-MLC2 beta gal infection show significant transcription only in cells derived from the cardiac muscle and not from the skeletal muscle. Quantitative analysis of the beta-galactosidase activity in a number of cell lines also confirms this result. The level of beta-galactosidase expression in rat neonatal cardiomyocytes infected with Ad-MLC2 beta gal is 8% of that found when primary cells are infected with Ad-RSV beta gal (containing a beta-galactosidase gene under the control of the Rous sarcoma virus promoter). The cardiomyocytes-specific expression is also found after injection of Ad-MLC2 beta gal directly into the rat myocardium, although the viral genome can be detected by polymerase chain reaction (PCR) in other tissues. Lack of expression after direct injection into liver and skeletal muscle confirms these results. The use of a tissue-specific promoter is a first step to restrict transgene expression to a particular cell type of the targeted tissue.