MHC class II+ human T-cell clones are able to simultaneously present and respond to peptide Ag and superantigen resulting in both proliferation and subsequent anergy. A major question remains as to whether a single T cell can present to itself or whether T-T cell interactions are required. We have employed a novel technique for inhibiting cell-to-cell contact that encapsulates individual T cells in agarose gel microdrops. Myelin basic protein-reactive individual CD4+ T-cell clones entrapped within these microdrops neither proliferated nor became anergized to either peptide Ag or Staphylococcal enterotoxin B (SEB), suggesting that cell-to-cell contact was required for T-cell presentation of Ag leading to proliferation and anergy. PMA treatment induced T-cell migration out of gel microdrops, restoring cell-to-cell contact and resulting in proliferation and anergy after T-cell coculture with peptide or superantigen. However, analysis of [Ca+2]i release revealed differences in T-cell responses to SEB versus peptide Ag. The addition of SEB, but not peptide Ag, induced a calcium flux in solitary T cells. Additionally, alpha HLA-DR mAb blocked peptide but not SEB-induced proliferation and anergy induction. Thus, SEB generated an early signal in solitary T cells that may not be a result of self stimulation via MHC class II. However, subsequent cell-to-cell contact was required for proliferation and anergy induction by SEB. These results indicate that peptide Ag requires a MHC class II-dependent cell-to-cell interaction for calcium flux, proliferation, and anergy induction, whereas SEB requires a MHC class II independent cell-to-cell interaction for proliferation and anergy induction after a TCR-generated calcium flux.