Irradiated (800 rads) AKR mice received intravenous (i.v.) reconstitution with a mixture of B10.BR T-depleted bone marrow cells and spleen cells. Only in groups of mice treated additionally with i.v. cyclophosphamide (Cy; 150 mg/kg), 24 hr before transplantation, was long-term (> 60% at 50 days) survival seen. In mice receiving only irradiation all animals died by 30 days post-transplantation. Histological changes consistent with graft-versus-host disease (GVHD) were seen in the liver of reconstituted mice at 30 days, along with an organ-specific increase in V beta 3 T-cell receptor-positive (TCR+) cells. No such increase in V beta 3 TCR+ cells was seen in the spleen from the same mice. These data are consistent with a tissue antigen-driven expansion of V beta 3 TCR+ cells associated with GVHD in the liver in this model. When we analysed cytokine production in vitro from CD3+ cells restimulated with 'host' (AKR) antigen-presenting cells (APC), we found a transition in cytokine production from preferential synthesis of type-1 cytokines [interleukin-2 (IL-2) and interferon-gamma (IFN-gamma)] at early times (day 15) post-reconstitution to increased production of type-2 cytokines [IL-4, transforming growth factor-beta (TGF-beta) and IL-10] at later times (day 30) post-reconstitution in Cy-treated recipients. Animals not receiving Cy did not show this 'switch' in cytokine production at later time points. We have observed a similar polarization in cytokine production, along with increased graft survival, in recipients of vascularized and non-vascularized allografts after portal venous (p.v.), but not i.v., pretransplant donor-specific immunization. We next studied AKR mice receiving 800 rads and subsequently reconstituted with B10.BR stem cells via the p.v. route. Again these mice showed prolonged survival (> 50% at 50 days), with polarization to IL-4, IL-10 and TGF-beta on restimulation of CD3+ cells in vitro at 30 days post-transplant and increased V beta 3 TCR+ cells in the liver. Infusion of anti-IL-12 monoclonal antibodies into irradiated mice receiving i.v. cell reconstitution produced a similar pattern of changes to those seen after p.v. reconstitution, while a combination of anti-IL-10 and anti-TGF-beta monoclonal antibodies reversed the changes seen after p.v. reconstitution. These data are consistent with an important role for differential cytokine production in the regulation of GVHD following allogeneic bone marrow transplantation.