Cardiac tissue engineering will remain only a prospect unless large numbers of therapeutic cells can be provided, either from small samples of cardiac cells or from stem cell sources. In contrast to most adult cells, cardiomyocytes are terminally differentiated and cannot be expanded in culture. We explored the feasibility of enabling the in vitro expansion of primary neonatal rat cardiomyocytes by lentivector-mediated cell immortalization, and then reverting the phenotype of the expanded cells back to the cardiomyocyte state. Primary rat cardiomyocytes were transduced with simian virus 40 large T antigen (TAg), or with Bmi-1 followed by the human telomerase reverse transcriptase (hTERT) gene; the cells were expanded; and the transduced genes were removed by adenoviral vector expressing Cre recombinase. The TAg gene was more efficient in cell transduction than the Bmi-1/hTERT gene, based on the rate of cell proliferation. Immortalized cells exhibited the morphological features of dedifferentiation (increased vimentin expression, and reduced expression of troponin I and Nkx2.5) along with the continued expression of cardiac markers (alpha-actin, connexin-43, and calcium transients). After the immortalization was reversed, cells returned to their differentiated state. This strategy for controlled expansion of primary cardiomyocytes by gene transfer has potential for providing large amounts of a patient's own cardiomyocytes for cell therapy, and the cardiomyocytes derived by this method could be a useful cellular model by which to study cardiogenesis.