Heart attacks and congestive heart failure remain among the world's most prominent health challenges despite the many breakthroughs. Bone marrow mesenchymal stem cells (BMSCs) have the potential to transdifferentiate into myocytes if an appropriate cardiac environment is provided. This study is meant to investigate the ability of BMSCs to differentiate into cardiomyocytes in a conditioned medium. BMSCs were isolated from rat femurs and tibias using Percoll gradient centrifugation method. Cells were expanded as undifferentiated cells in culture for more than 3 passages and their phenotypes were identified with flow cytometer. BMSCs were cocultured with neonatal rat ventricular myocytes in a rate of 1:10 separated by semipermeable membrane. BMSCs marker of CD29 were highly expressed (98.89+/-1.2%); however, CD34 could hardly be identified (5.61+/-0.1%). After coculturing with myocytes, some of BMSCs showed contraction which became more regular and more vigorous. As assessed by RT-PCR, SERCA2 and RyR(2) were expressed by newly formed cells from 1 to 3 weeks. Immunostaining of newly differentiated BMSCs revealed positivity for cTnT. Some of these cells were positive for sarcomeric alpha-actinin, desmin, cTnT, and cTnI. Western blotting showed that cTnI protein expression was upregulated in these cells from 1 to 3 weeks. Newly formed BMSCs exhibited ultrastructural features of sarcomere formation and inward rectifier potassium current (I(K1)). It is concluded that BMSCs possess the potential to differentiate into cardiomyocytes in the cardiac environment. BMSCs provide an excellent model for development of stem cell therapeutics, and their potential in the cardiac repair under various pathological conditions.