Mesenchymal stromal cells (MSC) are progenitors of mesenchymal tissues including bones. Irradiation can damage the osteogenic activity of human marrow by suppressing osteoblasts leading to post-irradiation osteoporosis. However, the effect of therapeutic irradiation on MSC remains unexplored. We investigated the effects of various doses of X-ray irradiation on human MSC (hMSC) by measuring its post-irradiation proliferation and differentiation activities. Standard immunophenotypes and differentiating functions of the MSC were determined. Irradiation inhibited proliferation of hMSC up to two wk post-irradiation but thereafter, those residual surviving cells regained their normal proliferation rate. Bone forming activity as reflected by alkaline phosphatase (ALP) and calcium deposition were both reduced in a dose-dependent fashion. Maximum suppressive effect on osteogenic activity was noted in MSC treated with high-dose irradiation (12 Gy). Adipocyte percentage was also reduced by 50% in cultures that received >4 Gy. Attempts to protect the irradiated cells with 1 microM all-trans retinoic acid did not show any beneficial effect on MSC proliferation and differentiation. The direct impairment of proliferation and osteogenic differentiation potential of MSC by irradiation may contribute partly to the post-transplant osteoporosis.