The murine CD44 receptor family is thought to be involved in a variety of lymphocyte functions, including lymphopoesis, lymphocyte homing and cell migration. Herein, we show that murine CD44 also plays a role as a co-stimulatory molecule for the activation of CD4+ T cells. Ligation of CD44 by mAb enhanced IL-2 production of long-term cultured, anti-CD3-stimulated Th1 cell lines. Moreover, anti-CD44 mAb synergized with anti-CD28 mAb in exerting this effect. A synergism of anti-CD28 and anti-CD44 mAb to co-stimulate IL-2 production was also observed in anti-CD3-triggered, freshly isolated splenic CD4+ T cells. Blocking experiments with cyclosporin A indicated that the intracellular pathways used by the CD28 and CD44 molecules appear to be different. In contrast to the effects on the IL-2 production of Th1 cells, neither anti-CD44 mAb alone nor the combination of anti-CD44 with anti-CD28 were able to induce proliferation of anti-CD3-triggered Th1 cells. In accordance, triggering of CD44 and/or CD28 by mAb was not sufficient to reverse the previously described 'proliferative block'. This term describes the unresponsiveness of Th1 cells against IL-2, which occurs when Th1 cells are triggered by anti-CD3 in the absence of co-signals. These data lead us to propose a model of Th1 cell activation which includes two functionally different types of co-signals: one for IL-2 production and a separate one for proliferation.