Human mast cells (MC) were examined for expression of MHC class II antigens and for their ability to activate CD4⁺ T cell hybridomas through presentation of superantigen (SAg). HMC‐1, a leukemic immature MC line expressing class II Ags, was shown to efficiently present the staphylococcal enterotoxin B (SEB) SAg to responding T cell hybridoma on treatment with interferon‐γ (IFN‐γ), which up‐regulated class II molecules. The study was then extended to human normal MC. Almost pure (>99%) cord blood‐derived MC (CBMC) were shown to express class II Ags (HLA‐Dr and HLA‐DQ) and CD80, which were up‐regulated by IFN‐γ treatment and, to a lesser extent, by interleukin‐4 (IL‐4) and granulocyte‐macrophage colony‐stimulating factor (GM‐CSF). CBMC directly activated CD4⁺ T cell hybridomas through presentation of SEB and TSST1 SAgs. The production of IL‐2 required a cell‐to‐cell contact between T cells and CBMC and it was inhibited by anti‐class II antibodies. Furthermore, an additional pretreatment of CBMC by IFN‐γ or GM‐CSF or IL‐4 had no effect on their presenting efficiency. This previously unknown function of human MC, i.e., MHC class II‐dependent activation of CD4⁺ T cells, may be critical in subsequent cellular activation events because colocalization of mast and T cells is frequently observed at sites of antigen entry. J. Leukoc. Biol. 66: 105–112; 1999.