gammadelta T cell populations are known to expand in response to intracellular bacterial infectious agents regardless of previous priming. We have shown previously that soluble factor(s) produced by Mycobacterium-stimulated monocytes activate cord blood gammadelta T cells to proliferate. In this study, we investigated whether cytokines produced by monocytes are responsible for gammadelta T cell activation in vitro: interleukin (IL)-1beta, IL-6, IL-8, IL-12, tumor necrosis factor (TNF)-alpha and granulocyte/macrophage colony-stimulating factor were examined. Recombinant human IL-12 stimulated gammadelta T cells, but not alphabeta T cells in peripheral blood mononuclear cells, to express CD25 on their surfaces, and to expand in number in vitro. IL-12-primed gammadelta T cell numbers increased to a greater extent in the culture to which exogenous IL-2 (5 U/ml) was added. Anti-TNF-alpha monoclonal antibody inhibited IL-12-induced up-regulation of CD25 on gammadelta T cells, suggesting that endogenous TNF-alpha may play a role in IL-12-induced activation of gammadelta T cells. Recombinant TNF-alpha synergistically augmented IL-12-induced activation of gammadelta T cells. Furthermore, IL-12 up-regulated TNF receptors on gammadelta T cells in vitro: TNF-alpha binding to its receptor induced CD25 expression on the gammadelta T cells in an autocrine or paracrine fashion, or perhaps both. It also became evident that both IL-12 and TNF-alpha were produced by mycobacterial lysate-stimulated monocytes. Taken together, these results suggest that upon confrontation with mycobacterial organisms, gammadelta T cells can be quickly and antigen-nonspecifically activated by soluble factors including IL-12 and TNF-alpha, both of which are produced by mononuclear phagocytes in response to mycobacterial organisms.