Anthracyclines, including doxorubicin, are widely used in the treatment of leukemia. While the effects of doxorubicin on hematopoietic cells have been characterized, less is known about the response of human mesenchymal stem cells (hMSCs) in the bone marrow stroma to anthracyclines. We characterized the effect of doxorubicin on key DNA damage responses in hMSCs, and compared doxorubicin sensitivity and DNA damage response activation between isolated hMSCs and the chronic myelogenous leukemia cell line, K562. Phosphorylation of H2AX, Chk1, and RPA2 was more strongly activated in K562 cells than in hMSCs, at equivalent doses of doxorubicin. hMSCs were relatively resistant to doxorubicin such that, following exposure to 15 mu M doxorubicin, the level of cleaved caspase-3 detected by western blotting was lower in hMSCs compared to K562 cells. Flow cytometric analysis of cell cycle progression demonstrated that exposure to doxorubicin induced G2/M phase arrest in hMSCs, while 48 h after exposure, 15.6 % of cells were apoptotic, as determined from the percentage of cells having sub-G1 DNA content. We also show that the doxorubicin sensitivity of hMSCs isolated from a healthy donor was comparable to that of hMSCs isolated from a chronic lymphocytic leukemia patient. Overall, our results demonstrate that high doses of doxorubicin induce the DNA damage response in hMSCs, and that cultured hMSCs are relatively resistant to doxorubicin.