Stimulation of IL-2-producing T cell clones with chemically modified APC and Ag induces a state of proliferative unresponsiveness, i.e., subsequent stimulation with normal APC and Ag fails to elicit IL-2 production. One possible effect of chemical modification on the APC is the destruction of its ability to provide costimulatory signals. To test this, various potential costimulators were added to T cells at the time of exposure to Ag and chemically modified APC. None of the cytokines tested, including IL-1, had a positive effect; however, addition of allogeneic spleen cells allowed a T cell proliferative response and prevented the induction of subsequent unresponsiveness. Fractionation of the spleen cells showed that low density B cells and macrophages were the best source of costimulatory activity. Allogeneic resting B cells provided some costimulatory activity and resting T cells, none at all. Attempts to mimic costimulatory signals with the phorbol ester PMA were only partially successful. PMA prevented the induction of T cell unresponsiveness but failed to allow T cell proliferation in response to Ag plus chemically modified APC. Our results suggest that IL-2 production by normal T cell clones is dependent not only on T cell receptor occupancy, but also on short range costimulatory signals that are provided to different degrees by various non-T accessory cells.