Stem cells derived from a patient's own heart offer a potentially attractive source for cellular transplantation therapy with the goal of myocardial regeneration. While endogenous myocardial repair capacity is limited, ex vivo expansion of resident stem cells, followed by delivery to the heart, may favor engraftment, regeneration and functional improvement. Percutaneous endomyocardial biopsy specimens from 70 adult patients grown in primary culture developed multi-cellular clusters known as cardiospheres, which were then plated to yield cardiosphere-derived cells (CDCs). Biopsy specimens (~20 mg on average) from 69 of 70 patients yielded cardiosphere-forming cells and generated 1.7±0.4 million CDCs within 45±7 days. Human cardiospheres and CDCs were characterized by their expression of myocyte and stem cell-related antigens (alpha-sarcomeric actin, connexin 43, Nkx2.5, c-Kit, CD105, CD90, MDR1, CD133, CD34, CD31, CD45) and possessed antigenic characteristics of stem cells at each stage of processing, as well as proteins vital for myocyte contractile and electrical function. Human and porcine CDCs co-cultured with neonatal rat ventricular myocytes cycled calcium in sync with neighboring myocytes, demonstrated nodal- and ventricular-like action potentials, exhibited endogenous inward sodium currents, inwardly rectifying potassium currents, and L-type calcium currents when transduced with the beta-subunit of the L-type calcium channel. Finally, human CDCs were injected into the border zone of acute myocardial infarcts in immunodeficient mice, with histological examination and echocardiographic left ventricular function as endpoints. CDCs engrafted and migrated into the infarct zone and surrounding viable tissue. After 20 days, the CDC-treated group had a higher percentage of viable myocardium within the infarct zone as compared to the fibroblast-treated control group (24.7±1.2% vs 16.4±1.9%, p<0.05) and a higher LVEF as compared to the fibroblast-treated group (38.8±1.7% vs 24.5±1.8%, p<0.05). CDCs within the viable myocardium could be found expressing alpha-sarcomeric actin and von Willebrand factor. CDCs are cardiogenic in vitro and produce cardiac regeneration and improve heart function in a mouse infarct model, motivating further development for therapeutic applications in patients.