Polysaccharides of pathogenic extracellular bacteria commonly have negatively charged groups or no charged groups at all. These molecules have been considered classic T cell-independent Ags that do not elicit cell-mediated immune responses in mice. However, bacterial polysaccharides with a zwitterionic charge motif (ZPSs), such as the capsular polysaccharides of many strains of Bacteroides fragilis, Staphylococcus aureus, and Streptococcus pneumoniae type 1 elicit potent CD4(+) T cell responses in vivo and in vitro. The cell-mediated response to ZPS depends on the presence of both positively charged and negatively charged groups on each repeating unit of the polysaccharide. In this study, we define some of the requirements for the presentation of ZPS to CD4(+) T cells. We provide evidence that direct interactions of T cells with APCs are essential for T cell activation by ZPS. Monocytes, dendritic cells, and B cells are all able to serve as APCs for ZPS-mediated T cell activation. APCs lacking MHC class II molecules do not support this activity. Furthermore, mAb to HLA-DR specifically blocks ZPS-mediated T cell activation, while mAbs to other MHC class II and class I molecules do not. Immunoprecipitation of lysates of MHC class II-expressing cells following incubation with ZPS shows binding of ZPS and HLA-DR. Electron microscopy reveals colocalization of ZPS with HLA-DR on the cell surface and in compartments of the endocytic pathway. These results indicate that MHC class II molecules expressing HLA-DR on professional APCs are required for ZPS-induced T cell activation. The implication is that binding of ZPS to HLA-DR may be required for T cell activation.