S keletal myoblast transplantation is a potential treatment for congestive heart failure. To study the functional activity of both donor and host myocytes following transplantation, skeletal myoblasts expressing an enhanced green fluorescent protein (EGFP) transgene were transplanted into hearts of nontransgenic recipients, and changes in intracellular calcium concentration ([Ca²⁺]_i) were monitored in donor and host cells. While the vast majority of donor-derived myocytes were observed to be functionally isolated from the host myocardium, a small population of donor myocytes exhibited action potential–induced calcium transients in synchrony with adjacent host cardiomyocytes. In many cases, the durations of these [Ca²⁺]_i transients were heterogeneous compared with those in neighboring host cardiomyocytes. In other studies, EGFP-expressing donor myoblasts were transplanted into the hearts of adult transgenic recipient mice expressing a cardiomyocyte-restricted β-gal reporter gene. A small population of myocytes was observed to express both reporter transgenes, indicating that the transplanted myoblasts fused with host cardiomyocytes at a very low frequency. These cells also expressed connexin43, a component of gap junctions. Thus engraftment of skeletal myoblasts generated spatial heterogeneity of [Ca²⁺]_i signaling at the myocardial/skeletal muscle interface, most likely as a consequence of fusion events between donor myoblasts and host cardiomyocytes.