JJ316 and JJ319 are rat CD28-specific monoclonal antibodies (mAb) of the gamma1 kappa isotype with identical co-stimulatory potency. At a concentration 100-1000-fold higher than that required for co-stimulation, JJ316, but not JJ319 induces massive proliferation of all T cell subsets in vitro without T cell receptor (TCR) triggering. "Direct" stimulation by JJ316 is fully blocked by JJ319, indicating that it is not due to cross-reactivity of JJ316 with the TCR complex or other activating receptors. JJ316 binds much more slowly to primary T cells than JJ319, whereas both antibodies bind with similar kinetics to CD28-transfected L-929 cells, suggesting that JJ316 binding to T cells requires redistribution or a conformational change of CD28. In vivo, JJ316 but not JJ319 induces rapid and transient proliferation of most CD4 T cells and, indirectly, of B cells. These data show that TCR engagement is not an absolute prerequisite either in vitro or in vivo for the induction of T cell proliferation through CD28 and suggest that mAb JJ316 is able to stimulate resting T cells directly by recruiting CD28 molecules from an inactive to an active form.