Co-stimulation via the CD28/CTLA-4 system appears critical for T cell proliferation to peptide antigens presented in association with MHC. In this study, we examine the roles of CD28 and CTLA-4 in the response of murine T cells to the superantigen staphylococcal enterotoxin B (SEB). In vitro, antibodies against B7-1/B7-2 or Fab fragments of anti-CD28 antibodies significantly inhibit the response of splenocytes to SEB. Conversely, Fab fragments of anti-CTLA-4 antibodies augment the proliferative response. Further, addition of blocking antibodies directed against B7-1/B7-2 augment proliferation co-stimulated by intact anti-CD28 antibodies. These data support the hypothesis that CD28 and CTLA-4 exert opposing effects upon early T cell activation. In vivo, Intact anti-CD28 antibodies and non-stimulatory Fab fragments of anti-CD28 appear to have similar inhibitory effects upon the expansion of V_β8⁺ T cells. In contrast, both intact and Fab fragments of anti-CTLA-4 appear to amplify this expansion. We conclude that the SEB response is significantly augmented by CD28-derived signaling and this in turn may be attenuated by signals through CTLA-4.