Cloned alpha beta TcR+ CD3+ CD4- CD8- T cells, with natural suppressor (NS) activity, were cocultured with thymocytes in the presence or absence of mitogen or cytokines. Whereas thymocytes show a minimal response to phytohemagglutinin (PHA), IL-2, or IL-4 alone, they proliferate vigorously when cocultured with irradiated cloned NS cells in the presence of PHA or IL-2 or IL-4, but not with IL-1, IL-3, IL-6, IL-7, IFN-gamma, or GM-CSF. Among a total of 11 NS cell clones, derived from the spleen or thymus, only one clone (NR-1) did not induce thymocyte activation in synergy with PHA. This costimulation is most likely mediated by soluble factor(s), since supernatants, obtained from NS cells activated with phorbol ester (PMA) and calcymicin (A23187) or with solid phase anti-CD3 mAb, enhance thymocyte DNA synthesis in the presence of a mixture of PHA, IL-2, and IL-4. The latter factor does not appear to be a previously described lymphokine, since PMA- and A23187-activated NS cells secrete IL-3, TGF-beta, IFN-gamma, GM-CSF, and TNF-alpha, but not IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, nor IL-10. None of the factors, identified in the NS cell supernatants, was able to stimulate thymocyte DNA synthesis. This study shows that, in addition to their previously reported suppressor function, cloned NS cells can exert immunostimulatory activities by virtue of a soluble mediator.