The induction of peripheral tolerance following oral antigen administration in several autoimmune disease and conventional animal models correlates with the production of transforming growth factor-beta (TGF-beta) and T helper type 2 (Th2) cytokines. The factors regulating TGF-beta production and its relation to the Th2 response, however, have not been defined. We demonstrate that the systemic administration of antibodies to interleukin (IL)-12 to ovalbumin (OVA)-T cell receptor (TCR) transgenic mice fed high doses of OVA, followed by systemic OVA challenge, substantially enhances TGF-beta, but not IL-4 production by peripheral T cells. Furthermore, we demonstrate in an in vitro T cell differentiation model that naive (CD4+/Mel-14hi) OVA-TCR-T cells stimulated with OVA-pulsed dendritic cells (DC) produce four- to fivefold higher amounts of TGF-beta when cultured with anti-IL-12 or anti-interferon-gamma (IFN-gamma). In this in vitro system, IL-4 was not required for TGF-beta production by T cells; however, it appeared to enhance levels of TGF-beta by promoting the growth of TGF-beta-producing cells. Our findings demonstrate that IL-12 and IFN-gamma are important negative regulators of TGF-beta production both in vivo and in vitro, and that their modulation may be of benefit for the treatment of autoimmune disorders.