T cell activation requires two signals: a signal from the TCR and a co-stimulatory signal provided by antigen-presenting cells (APC). In addition to CD28, multiple molecules on the T cell have been described to deliver co-stimulatory signals. Here, we investigated whether there exist quantitative or qualitative differences in the co-stimulatory capacity between CD28 and other molecules. Anti-CD28 monoclonal antibody (mAb) and mAb against CD5, CD9, CD2, CD44 or CD11a all induced activation of naive T cells in the absence of APC when co-immobilized with a submitogenic dose of anti-CD3 mAb. [3H]Thymidine incorporation determined 2 days after co-stimulation was all comparable. In contrast to progressive T cell proliferation induced by CD28 co-stimulation, co-stimulation by other T cell molecules led to a decrease in viable cell recovery along with the induction of apoptosis of once activated T cells. This was associated with a striking difference in IL-2 production; CD28 co-stimulation induced progressively increasing IL-2 production, whereas co-stimulation by other molecules produced limited amounts of IL-2. Addition of recombinant IL-2 to the latter cultures corrected the induction of apoptosis, resulting in levels of cellular proliferation comparable to those observed for CD28 co-stimulation. These results indicate that a fundamental difference exists in the nature of co-stimulation between CD28 and other molecules, which can be evaluated by the levels of IL-2 production, but not simply by [3H]thymidine incorporation.