Pre--CFU-S are characterized by their ability to generate spleen colony- forming cells (CFU-S) and by their ability to repopulate the hematopoietic system after damage. We have investigated their response to three consecutive injections of cytosine arabinoside (ara-C), given at t = 0, 12, and 20 hours. Nine hours after treatment, the number of CFU-S and pre--CFU-S was reduced to 10% or 30%, respectively. No pre-- CFU-S were in S-phase at this time, indicating that the pre--CFU-S losses were not caused by direct drug killing. Up to 1 year after treatment, pre--CFU-S were still depleted to 10% of normal, indicating that their proliferative quiescence was permanent. We have previously shown that inhibition of CFU-S recruitment with pGlu-Glu-Asp-Cys-Lys (pEEDCK) makes them ara-C resistant and prevents their decimation. We now found that this also prevented the excessive drainage of the pre-- CFU-S pool, suggesting that pre--CFU-S allocation into active hematopoiesis is triggered by the CFU-S deficit. pEEDCK may thus be applicable as a protector of the hematopoietic repopulation potential against cytostatic drug-induced aplasia. Postchemotherapeutic stimulator treatment with (pEEDCK)2-dimer did not ameliorate pre--CFU-S losses. Long-term culture-initiating cells (LTC-ICs) showed a similar pattern of irreversible reduction after cytostatic drug treatment, which could be prevented by pEEDCK. Our results suggest, that certain subclasses of hematopoietic stem cells (pre--CFU-S) are permanently quiescent and exhaustible and that the capacity for self-renewal is not a necessary property of all stem cell-like cells.