Prior in vitro studies suggested that different types of hematopoietic stem cells may differentiate into cardiomyocytes. The present work examined whether human CD34⁺ cells from the human umbilical cord blood (hUCB), cocultured with neonatal mouse cardiomyocytes, acquire the functional properties of myocardial cells and express human cardiac genes. hUCB CD34⁺ cells were cocultured onto cardiomyocytes following infection with a lentivirus encoding enhanced green fluorescent protein (EGFP). After 7 days mononucleated EGFP⁺ cells were tested for their electrophysiologic features by patch clamp and for cytosolic [Ca²⁺] ([Ca²⁺]_i) homeostasis by [Ca²⁺]_i imaging of X-rhod1 loaded cells. Human Nkx2.5 and GATA4 expression was examined in cocultured cell populations by Real Time (RT)-PCR . EGFP⁺ cells were connected to surrounding cells by gap junctions, acquired electrophysiologic properties similar to those of cardiomyocytes and showed action potential-associated [Ca²⁺]_i transients. These cells also exhibited spontaneous sarcoplasmic reticulum [Ca²⁺]_i oscillations and the associated membrane potential depolarization. However, RT-PCR of both cell populations showed no upregulation of human-specific cardiac genes. In conclusion, under our experimental conditions, hUCB CD34⁺ cells cocultured with murine cardiomyocytes formed cells which exhibited excitation-contraction coupling features similar to those of cardiomyocytes. However, expression of human specific cardiac genes was undetectable by RT-PCR.