We previously reported that murine bone marrow cells activated by interleukin-3 (IL-3) or granu- locyte-macrophage colony-stimulating factor (GM-CSF) had potent nonspecific natural suppressor (NS) cell activity. In the present study, we demonstrated that these activated NS cells released a soluble factor (or factors) capable of nonspecifically inhibiting T cell mitogenic responses. Consistent with the properties of Transforming growth factor-β (7GF-β), treatment of the NS supernates with heat failed to denature the factor, and in fact significantly increased its suppressive activity. The NS suppressor factor strongly inhibited proliferation of the TGF- β-sensitive tumor cell line, A549. Cytokine activation of suppressive activity correlated with the production of a 10- to 13-kDa protein, consistent with the size of TGF-β and rIL-3 induced a sevenfold increase in TGF-β transcription. Finally, neutralizing anti-TGF-β antibody inhibited the suppressive activity of the supernates, indicating that TGF-β was responsible for most, if not all, of the suppression expressed by these bone marrow NS cells.