We investigated the involvement of NF-kappaB/Rel transcription factors that reportedly can inhibit apoptosis in various cell types in the antiapoptotic mechanism of the cytoprotectant amifostine. In the nontumorigenic murine myeloid progenitor 32D cells incubated with amifostine, we detected a reduction of the IkappaBalpha cytoplasmic levels by Western blotting and a raising of nuclear NF-kappaB/Rel complexes by electrophoretic mobility shift assay. Amifostine inhibited by more than 30% the growth factor deprivation-induced apoptosis, whereas its effect failed when we blocked the NF-kappaB/Rel activity with an NF-kappaB/Rel-binding phosphorothioate decoy oligodeoxynucleotide. In human cord blood CD34(+) cells, the NF-kappaB/Rel p65 subunit was detectable (using immunofluorescence analysis) mainly in the cytoplasm in the absence of amifostine, whereas its presence was appreciable in the nuclei of cells incubated with the cytoprotectant. In 4 CD34(+) samples incubated for 3 days in cytokine-deficient conditions, cell apoptosis was reduced by more than 30% in the presence of amifostine (or amifostine plus a control oligo); the effect of amifostine was abolished in cultures with the decoy oligo. These findings indicate that the inhibition of hematopoietic progenitor cell apoptosis by amifostine requires the induction of NF-kappaB/Rel factors and that the latter can therefore exert an antiapoptotic activity in the hematopoietic progenitor cell compartment. Furthermore, the identification of this specific mechanism underlying the survival-promoting activity of amifostine lends support to the possible use of this agent in apoptosis-related pathologies, such as myelodysplasias.