BACKGROUND: Mesenchymal stem cells (MSCs) that are capable of extensive self renewal and differentiation have attracted great attention as a promising tool for regenerative medicine and tissue engineering. Although MSCs can be isolated from various tissues, bone marrow currently represents one of the most reliable sources for providing a sufficient yield of cells in a good quality. Herein, used bone marrow processing kits were evaluated as a valuable source of MSCs. METHODS: Bone marrow mononuclear cells (MNCs) were recovered from used bone marrow processing kits after routine bone marrow processing by using the COBE 2991 Cell Processor (CaridianBCT Inc.). The MSCs were isolated from the recovered MNCs using a standard plastic adherence method. Immunophenotyping and differentiation assays were performed to clarify the characteristics of the isolated MSCs. RESULTS: An average of 1x108 bone marrow MNCs was collected, and the MSCs were successfully isolated from the recovered bone marrow MNCs in all case. The isolated MSCs were positive for essential MSC surface molecules (CD29, CD44, CD73, CD90, CD105) and they were negative for most hematopoietic and endothelial cell markers (CD34, CD45, CD31, CD14). The isolated MSCs were capable of differentiation along the osteogenic, adipogenic and chondrogenic pathways. CONCLUSION: MSCs isolated from used bone marrow processing kits are an alternative and ethical source of bone marrow derived MSCs, and they can be used for research purposes.