The 39-kDa V antigen (Vag) of pathogenic Yersinia species has been described to be a potent suppressor of TNF production. The underlying cellular and molecular mechanisms, however, are completely undefined. Here we show that Vag does not act directly on macrophages, the primary source of TNF, but rather requires help of activated T cells for TNF suppression. Suppression of TNF strictly required the presence of T cell stimuli, i.e. concanavalin A or immobilized anti-CD3 antibody. In controls, suppression of TNF was completely blocked by anti-recombinant polyhistidine V antigen (rVagHis) IgG. As determined by transwell chamber experiments, suppression of TNF by rVagHis did not depend on cell-to-cell contact, indicating that it is mediated by an as yet unknown soluble factor. This is the first report to show a suppressive effect of rVagHis on TNF production in tissue culture. The results demonstrate the importance of activated T cells for suppression of TNF expression by rVagHis in vitro.