Gamma-irradiated ex vivo bovine monocytes induce proliferation of gamma/delta T cells in the autologous mixed lymphocyte reaction (AMLR), whereas when not irradiated they prevent this response. In contrast, non-irradiated autologous monocytes have no effect on bovine alpha/beta T-cell proliferation in the allogenic MLR suggesting that the regulation is specific for gamma/delta T-cell responses. Here, we showed that the inhibition was not mediated by inducing cell death and that the ability of ex vivo monocytes to prevent proliferation of gamma/delta T cells was not generalized in that gamma/delta T cells still responded to mitogenic stimulation. Inhibition of the AMLR by non-irradiated monocytes could not be overcome by addition of interleukin-2 to the cultures or by costimulation with antibodies to WC1, a gamma/delta T-cell-specific cell-surface differentiation antigen shown elsewhere by us to be involved in activation of gamma/delta T cells. Furthermore, we showed that monocytes inhibited gamma/delta T-cell responses via a soluble product since inhibition occurred even when monocytes and gamma/delta T cells were separated by membranes of transwells or when supernatants from monocyte cultures were added to AMLR cultures. Maximal secretion of the inhibitory product by the monocytes occurred during the first 6 hr of in vitro culture at 37 degrees, rapidly decreased thereafter, and did not occur when monocytes were incubated at 4 degrees. The inhibition was not attributable to nitric oxide, reactive oxygen intermediates, prostaglandin E2 or transforming growth factor-beta (TGF-beta) but the ability of monocyte supernatants to mediate inhibition was sensitive to heating at 65 degrees. Lipopolysaccharide and granulocyte-macrophage colony-stimulating factor activation of monocytes temporarily abrogated their ability to inhibit proliferation. In contrast, heat-shocking had no effect on their ability to inhibit. We hypothesize that non-irradiated monocytes produce the inhibitory material in vivo in order to regulate gamma/delta T-cell responses to self-derived monocyte membrane components, but that when monocytes are altered by infection, transformation, irradiation, or cytokine activation, production of the inhibitor is temporarily suspended allowing stimulation of gamma/delta T cells to occur.