In the present work, we explored the cytokine-dependent regulation of bone marrow-derived mast cell (BMMC) antigen-presenting cell (APC) function, and co-stimulation requirements, and analyzed the nature of antigens presented to T cells. We observed an up-regulation of the APC function of mast cells induced by granulocyte/macrophage-colony-stimulating factor (GM-CSF) and a complete abrogation by interferon (IFN)-γ. Expression of co-stimulatory molecules CD80 and CD86 was suggested by the ability of mast cells to activate purified lymph node-derived T cells. Indeed, addition of the fusion protein mCTLA4-Ig strongly inhibited antigen presentation by mast cells to normal T cells and to the T cell hybridoma 3DO-54.8. The regulatory mechanisms of APC function by GM-CSF and IFN-γ were investigated by measuring CD80 and CD86 transcripts in mast cells. GM-CSF-treated mast cells showed a strong increase in the expression of both CD80 and CD86 transcripts, whereas in IFN-γ-treated mast cells, this expression was completely abrogated. Thus, up- and down-regulation of CD80 and CD86 expression by GM-CSF and IFN-γ is directly correlated to the APC function. In addition, we analyzed antigen presentation by mast cells of endogenous self-antigens. Mast cells failed to activate anti-I-A or anti-I-E-specific T cell hybridomas and alloreactive T cells in primary mixed lymphocyte reactions (MLR). Furthermore, mast cells did not present the mouse β2-microglobulin (mβ2-m) peptide 25–40, constitutively expressed on B cells. However, mast cells, especially those treated with GM-CSF, activated an anti-mβ2-m-specific T cell hybridoma in the presence of exogenous peptide. The minor lymphocyte-stimulating antigen-1 Mls-1a is a viral superantigen (vSAG) encoded by the the mouse mammary tumor provirus-7 (MMTV-7). Mast cells, despite a reasonable amount of major histocompatibility complex class II on the cell surface and the presence of MMTV transcripts predicted to encode the vSAG, cannot stimulate in vivo or in vitro Vβ6+ T cells specific for Mls-1a. In contrast, mast cells could present the exogenous bacterial SAG, staphylococcal enterotoxin B (SEB), to specific Vβ8+ T cells. The selective ability of mast cells to present exogenous antigens may have physiological relevance in that mast cells could participate in immune response regulatory mechanisms by discriminating self from nonself.