The staphylococcal exotoxin toxic shock syndrome toxin-1 (TSST-1) has been demonstrated to bind to monomorphic determinants on MHC-class II molecules. In this study, we have used TSST-1 to probe the role of MHC-class II molecules in the activation and differentiation of resting human B cells. Highly purified B cells were stimulated with TSST-1, alone or in combination with PMA or with anti-human IgM antibodies (anti-mu) and the resulting B cell proliferation and Ig production were monitored. On its own, TSST-1 failed to induce B cell proliferation or Ig production. However, TSST-1 synergized with PMA and with anti-mu in inducing B cell proliferation in the absence of any added T cells or T cell factors. TSST-1 did not induce or potentiate early activation events associated with anti-mu treatment such as phosphoinositide hydrolysis and Ca2+ mobilization. Also, TSST-1 did not potentiate the capacity of anti-mu to induce the transcription of early activation genes such as c-myc. Finally, in contrast to its capacity to promote mitogen-trigered B cell proliferation, TSST-1 failed to induce the differentiation of B lymphocytes into Ig-secreting cells in the absence of added T cells. These results indicate that TSST-1 delivers a comitogenic signal to B cells via MHC-class II molecules that is distinct from signals delivered via surface mu and further strengthens the role of MHC-class II molecules as signal transducing structures.