Transforming growth factor beta (TGF beta)-treated antigen-presenting cells (APC) pulsed with antigen induce tolerance in mice, i.e. inhibition of IFN-gamma production and delayed type hypersensitivity response. Although evidence suggests that regulatory T cells are involved, their mechanism of action is currently unknown and is the subject of the present study. Both CD4 and CD8 splenic T cells from mice injected i.v. with adherent thioglycolate-elicited peritoneal exudate cells cultured with TGF beta(2) and antigen (TGF beta-treated APC) transferred tolerance to naive recipients. Interestingly, TGF beta-treated APC from class II knockout mice were unable to induce tolerance in wild-type mice, whereas wild-type TGF beta-treated APC could induce tolerance in CD8 knockout mice. TGF beta was detected in cultures of lymphoid cells from mice injected with TGF beta-treated APC, and treatment with anti-TGF beta antibody in vivo impaired tolerance induction. TGF beta appeared to be involved in both the development of CD4 regulatory T cells and the effector function of the CD4 regulatory T cells. In summary, the important findings in this study are that CD4, and not CD8, regulatory T cells are required for tolerance induced by TGF beta-treated APC in naive mice, and tolerance appears to be mediated by a mechanism involving TGF beta.