Using a DNP-specific, class II-restricted T-cell line C9, we asked whether normal spleen cells directly labeled with DNFB (DNP-SC) can stimulate DNP-specific T cells. C9 cells were established from BALB/c mice primed with syngeneic DNP-SC subcutaneously; they proliferated and produced IL-2 and MIF in a DNP-specific, I-A-restricted manner. We found that syngeneic DNP-SC alone, either unfixed or fixed with gluteraldehyde, could not stimulate C9 cells. However, when DNP-SC were added to cultures of C9 cells plus syngeneic fillers, but not allogeneic fillers, potent stimulation occurred. Allogeneic DNP-SC were also stimulatory provided the cultures contained filler cells syngeneic to the C9 responding T cells. DNP-protein conjugates, however, did not induce stimulation, indicating that the T cells are DNP specific but not hapten specific. Overnight coculture of DNP-SC and irradiated normal spleen cells also produced potent stimulator cells. However, generation of these stimulator cells was inhibited by addition of chloroquine to the culture medium. These findings indicate that syngeneic filler cells acquire DNP from the DNP-SC and re-present the hapten to the T cells in the context of IA. This process appears to require antigen processing by the filler cells. Collectively the results indicate that labeling of cell membranes with reactive haptens may not directly produce an immunogenic complex which is recognized by T cells.