Stem cell therapy represents a potential novel additional therapy for acute myocardial infarction. Cardiac applications of stem cell therapy are now undergoing clinical trials though many properties, including localization, possible adhesion, and infiltration of the injected stem cells in the myocardium, have not been studied in detail even in vitro. To study these mechanisms in a controlled microenvironment, we developed a model where mesenchymal stem cells (MSCs) were transported into live, cultured cardiac explants for further co-culture. About 10×10(3) porcine MSCs were injected into freshly excised and isolated cardiac explants of the pig. The explants were present in the culture medium for up to 7 days, with the time course of viability of the myocardial tissue, and the migration and the localization of the injected MSCs were analyzed with histological and immunohistological stainings. The myocyte structure was observed to be well preserved, and proliferation of capillaries and myofibroblasts was detected at the explant periphery. There were injected MSCs localized in the capillaries and in contact with the endothelial cells. The migration range and the number of adherent MSCs increased over time, suggesting active movement of MSCs in the explant. Our results suggest that this cardiac explant culture model is a feasible method for studying the effects of stem cells in the myocardium in vitro.