JJ316 and JJ319 are rat CD28‐specific monoclonal antibodies (mAb) of the γ1χ 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.