Purified CD4+ T cells require TCR engagement and Ag-nonspecific co-stimulatory signals to produce IL-2 and proliferate. A number of recent studies have demonstrated that the interaction of the B7 molecule expressed on APC with the T cell-associated CD28 molecule provides a potent co-stimulatory signal to both freshly isolated CD4+ T cells and cloned Th1 cells. Earlier reports have described the role of cytokines, in particular IL-6 and IL-1, as costimulatory molecules for T cell activation. We previously reported that IL-6 and IL-1 synergize to co-stimulate proliferation of purified mouse CD4+ T cells in conjunction with anti-TCR mAb. In this report we explore the interaction of IL-6, IL-1, and CD28 signaling in the activation of mouse CD4+ T cells, and demonstrate that the co-stimulatory requirements of the cells vary depending on the mode of TCR stimulation. CD28 signaling is not sufficient to co-stimulate responses of high buoyant density CD4+ T cells to anti-TCR-conjugated agarose beads; there is an additional requirement that can be supplied by exogenous IL-6 but not by IL-1. In contrast, in responses to anti-TCR mAb that is passively bound to the bottom of culture wells, CD28 stimulation is sufficient to co-stimulate proliferation, resulting in a very high level of IL-2 production; there is no additional requirement for exogenous IL-6 or IL-1. Possible explanations for the differential requirement for IL-6 in the two systems are discussed. Our results are consistent with the notion that CD28 signaling plays a central role in co-stimulating T cell responses. However, the results also suggest that, depending on the nature of the TCR stimulus, T cell activation may also require additional co-stimulatory signals provided by cytokines.