Islet-1 expression identifies populations of progenitor cells in embryonic, fetal, and newborn murine hearts that are able to give rise to all cardiac cell lineages ex vivo and in vivo. Using systematic immunohistochemistry, we investigated whether islet-1-positive cells are present in adult mouse heart from the perspective of their potential therapeutic utility. The presence, localization, and nature of islet-1-positive cells were assessed in mice of different strains, ages, and conditions. Islet-1-positive cells were present in mouse heart from postnatal day 1 to young adulthood. Depending on the strain, these cells were organized in either 1 or 2 types of clusters localized to restricted areas, at a distance of 6%-35% of the heart length from the base. The first type of cluster was present in all strains and consisted of neural crest-derived cells that formed cardiac ganglia. The number of cells remained stable (a few hundred) from neonatal up to adult ages, and variations were noted between strains regarding their long-term persistency. The second type of cluster was essentially present in 129SvJ or Balb/C strains and absent from the other strains tested (C57BL/6J, C3H, SJL). It consisted of cells expressing highly ordered sarcomeric actin, consistent with their having cardiomyocyte identity. These cells disappeared in animals older than 4 months. Neither the number nor the type of islet-1-positive cells varied with time in a mouse model of dilated cardiomyopathy. Our studies demonstrate that islet-1-positive cells are relatively few in number in adult murine heart, being localized in restricted and rather inaccessible areas, and can represent both neural crest and cardiomyocyte lineages.