We have previously shown that dendritic cells isolated after overnight culture, which can express B7 and are potent stimulators of naive T cell proliferation, are relatively poor at inducing the proliferation of a panel of murine T helper 1 (Th1) clones. Maximal stimulation of Th1 clones was achieved using unseparated splenic antigen presenting cells (APC). An explanation for these findings is provided in the present study where we show that FcR+ L cells transfected with B7 stimulate minimal proliferation of Th1 clones in response to anti-CD3 antibodies, in contrast to induction of significant proliferation of naive T cells. However, addition of interleukin 12 (IL-12) to cultures of Th1 cells stimulated with anti-CD3 and FcR+ B7 transfectants resulted in a very pronounced increase in proliferation and interferon γ (IFN-γ) production. Exogenous IL-12 did not affect the B7- induced proliferation of naive T cells. This showed that whereas costimulatory signals delivered via B7-CD28 interaction are sufficient to induce significant proliferation of naive T cells activated through occupancy of the T cell receptor, Th1 T cell clones require cooperative costimulation by B7 and IL-12. This costimulation was shown to be specific by inhibition of proliferation and IFN-γ production using chimeric soluble cytolytic T lymphocyte-associated antigen 4-human IgG1Fc (CTLA4-Ig) and anti-IL-12 antibodies. Furthermore, the significant antigen specific proliferation and IFN-γ production by Th1 clones observed when splenocytes were used as APC was almost completely abrogated using CTLA4-Ig and anti-IL-12 antibodies. Thus two costimulatory signals, B7 and IL-12, account for the ability of splenic APC to induce maximal stimulation of Th1 clones. IL-10 downregulates the expression of IL-12 by IFN-γ-stimulated macrophages and this may account largely for the ability of IL-10 to inhibit APC function of splenic and macrophage APC for the induction of Th1 cell proliferation and IFN-γ production. Indeed we show that IL-12 can overcome the inhibitory effect of IL-10 for the APC-dependent induction of proliferation and IFN-γ production by Th1 clones. These results suggest that proliferation by terminally differentiated Th1 clones, in contrast to naive T cells, requires stimulation via membrane-bound B7 and a cytokine, IL-12. It is possible that these signals may result in the activation of unresponsive T cells during an inflammatory response. IL-10, by its role in regulating such innate inflammatory responses, may thus help to maintain these T cells in an unresponsive state.