We describe the effects of 4-hydroperoxycyclophosphamide (4-HC) on the hematopoietic and stromal elements of human bone marrow. Marrow cells were exposed to 4-HC and then assayed for mixed (CFU-Mix), erythroid (BFU-E), granulomonocytic (CFU-GM), and marrow fibroblast (CFU-F) colony-forming cells and studied in the long-term marrow culture (LTMC) system. The inhibition of colony formation by 4-HC was dose and cell-concentration dependent. The cell most sensitive to 4-HC was CFU-Mix (ID50 31 mumol/L) followed by BFU-E (ID50 41 mumol/L), CFU-GM (ID50 89 mumol/L), and CFU-F (ID50 235 mumol/L). In LTMC, a dose-related inhibition of CFU-GM production was noted. Marrows treated with 300 mumol/L 4-HC were completely depleted of CFU-GM but were able to generate these progenitors in LTMC. Marrow stromal progenitors giving rise to stromal layers in LTMC, although less sensitive to 4-HC cytotoxicity, were damaged by 4-HC also in a dose-related manner. Marrows treated with 4-HC up to 300 mumol/L, gave rise to stromal layers composed of fibroblasts, endothelial cells, adipocytes, and macrophages. Cocultivation experiments with freshly isolated autologous hematopoietic cells showed that stromal layers derived from 4-HC-treated marrows were capable of sustaining the long-term production of CFU-GM as well as controls. CONCLUSIONS (1) Hematopoietic progenitors cells, CFU-Mix, BFU-E, and CFU-GM, are highly sensitive to 4-HC, whereas marrow stromal progenitor cells are relatively resistant. (2) Marrows treated with 300 mumol/L 4-HC that are depleted of CFU-Mix, BFU-E, and CFU-GM can generate CFU-GM in LTMC, suggesting that most primitive hematopoietic stem cells (not represented by CFU-Mix) are spared by 4-HC up to this dose. (3) Consequently, the above colony assays are not suitable tools for predicting pluripotent stem cell survival after 4-HC treatment in vitro.