Cell therapy techniques are a promising option for tissue regeneration; especially in cases such as heart failure where transplantation is limited by donor availability. Multiple cell types have been examined for myocardial regeneration, including mesenchymal stem cells (and other bone marrow-derived cells), induced pluripotent stem cells, embryonic stem cells, cardiosphere-derived cells, and cardiac progenitor cells (CPCs). CPCs are multipotent and clonogenic, can be harvested from mature tissue, and have the distinct advantages of autologous transplant and lack of tumor formation in a clinical setting. Here we focus on the isolation, expansion, and myocardial differentiation of rat CPCs. Brief adaptations of the protocol for isolation from mouse and human tissue are also provided.