Mesenchymal stem cells (MSCs) are an important cellular component of the bone marrow microenvironment for supporting haemopoiesis. However, their response to high-dose chemotherapy remains unknown. We assessed the acute direct effects of individual chemotherapeutic agents on human MSCs (hMSCs). Using an in vitro culture system, the chemosensitivity of hMSCs was determined by XTT (2,3-bis(2-methoxy-4-nitro-5-sulphophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide) assay in comparison with that of NB-4 cells, a leukaemic cell line, and normal peripheral blood mononuclear cells. The recovery of cell numbers following exposure to chemotherapeutic agents and chemotherapy-induced apoptosis of hMSCs were evaluated. Human MSCs were resistant to chemotherapeutic agents commonly used in bone marrow transplantation (BMT) (i.e. busulphan, cyclophosphamide and methotrexate). However, they were relatively sensitive to a panel of cytotoxic agents, such as paclitaxel, vincristine, etoposide and cytarabine. Furthermore, different recovery patterns were noted. There was sustained suppression in hMSCs following 3 d exposure to paclitaxel, cytarabine and etoposide. In contrast, significant recovery was seen in hMSCs treated with dexamethasone and vincristine respectively. Human MSCs have different patterns of response to a panel of chemotherapeutic agents commonly used in BMT or cancer therapy. Understanding this variation is important in optimizing conditioning regimens for BMT.