In the last few years, a major goal of cardiac research has been to drive stem cell differentiation to replace damaged myocardium. Several research groups have attempted to differentiate potential cardiac stem cells (CSCs) using bi- or three-dimensional systems supplemented with growth factors or molecules acting as differentiating substances. We hypothesize that these systems failed to induce a complete differentiation because they lacked an architectural space. In the present study, we isolated a pool of small proliferating and fibroblast-like cells from adult rat myocardium. The phenotype of these cells was assessed and the characterized cells were cultured in a collagen I/OPLA scaffold with horse serum to obtain fine myocardial differentiation. C-KitPOS/Sca-1POS CSCs fully differentiated in vitro when an environment more similar to the CSC niche was created. These experiments demonstrated an important model for the study of the biology of CSCs and the biochemical pathways that lead to myocardial differentiation. The results pave the way for a new surgical approach. J. Cell. Physiol. 221: 729–739, 2009. © 2009 Wiley-Liss, Inc.