Tissue and marrow (BM) in vivo O2 tensions have been estimated at 23 to 40 mm Hg (3% to 5% O2). We have investigated cellular regulation of burst-forming units-erythroid (BFU-E) under 5% O2. BFU-E from BM mononuclear cells (MNC) were cultured in methylcellulose medium and erythropoietin (Ep) +/- monocyte-conditioned medium (MoCM, a source of burst-promoting activity, BPA) in the presence or absence of autologous T cells (T) and/or monocytes (M phi) under either 5% or 21% O2 after depletion of T (MNC-T), M phi (nonadherent buoyant cells, NAB) or both T and M phi depletion (NAB-T). MNC BFU-E growth under 5% O2 was augmented over 0.1 to 1.5 U/mL of Ep. BFU-E augmentation under 5% O2 was abolished by depletion of BM M phi, T, or both from MNC. The addition of MoCM affected neither a BFU-E increase under 5% O2 nor the abrogation of that increase upon T or M phi depletion. The addition of 5% to 20% M phi or 10% to 20% T to NAB-T failed to restore the BFU-E increase under 5% O2. However, BFU-E augmentation under 5% O2 was reestablished when 10% autologous M phi, 10% T, or 10% T plus 10% M phi were added back to marrow NAB, MNC-T, or NAB-T. BM BFU-E was not augmented in the presence of varying concentrations of catalase, superoxide dismutase, or reduced glutathione at 21% O2; moreover, BFU-E augmentation was maintained at 5% O2 relative to 21% O2 in the presence of each of these antioxidants. CM prepared under 5% or 21% O2 from BM M phi, T, or M phi plus T were assessed for BPA against BM NAB-T using a sensitive BPA assay incorporating delayed Ep addition to cultures. Only CM from mixtures of M phi and T cells at 5% O2 demonstrated potent BPA; little or no BPA was detected with T or M phi CM at 5% O2 and at 21% O2 or T and M phi CM at 21% O2. The sensitivity of NAB-T BFU-E to exogenous BPA was virtually identical at 21% and 5% O2. These results indicate that human BM BFU-E are augmented under 5% O2 and that T cells and M phi together mediate that augmentation by collaborating to produce BPA-like activity in response to physiological O2 tensions.