Bacterial superantigens can bind TCR in the absence of MHC class II molecules and activate T lymphocytes when cocultured with certain class II- deficient accessory cells. It has not been determined, however, whether these accessory cells provide direct costimulation to the T cell or serve to present superantigens via a nonconventional ligand. We have identified a human adenocarcinoma cell line, SW480, that assists in the activation of human T cells by the staphylococcal enterotoxins B (SEB), C1 (SEC1), and D (SED), but not SEA, SEC2, SEC3, or SEE. SW480 cells did not express class II molecules, and anti-class II mAbs did not inhibit T cell proliferation, supporting the hypothesis that class II is not absolutely required for enterotoxin-mediated T cell activation. The TCR Vβ profile of T cells stimulated by SEB plus SW480 cells was similar to that of T cells stimulated by SEB plus class II+ APC, indicating that TCR-SEB interactions were preserved in the absence of class II molecules. Binding studies failed to detect specific association of SEB with SW480 cells, suggesting that SW480 cells do not express receptors for enterotoxin. SEB coupled to beads, however, stimulated T cell proliferation, but only in the presence of SW480 cells. SW480 cells express both ICAM-1 and LFA-3 molecules, and the addition of Abs to these receptors inhibited T cell proliferation. These findings support a model in which certain enterotoxins engage the TCR independent of MHC class II or other specific presenting molecules and induce T cell proliferation with signals provided by nonconventional accessory cells.