In this study, our aim was to develop a new simple technique for isolation of mesenchymal stem cells from adipose tissue. For this purpose, mesenchymal stem cells were isolated from rat adipose tissue by using the primary explant culture technique. When the cells became confluent, they were passaged 4 times by using the standard trypsinization method with trypsin/EDTA solution. Cells at second passage were characterized by using immunofluorescence staining against CD13 and CD29 markers. The results showed that these cultured cells were positive for CD13 and CD29 markers. Flow cytometry analysis was also done against CD29, CD90, CD54, MHC Class I, CD45, CD 106, and MHC Class II for characterization of mesenchymal stem cells. The results of flow cytometry analysis showed that these cells were mesenchymal stem cells. Half of the cells were cryopreserved at all passages for future applications. It is thought that these mesenchymal stem cells can be used in therapy of cardiovascular diseases as an alternative technique in the near future.