We have examined the effectiveness of a bacterial superantigen, staphylococcal enterotoxin B (SEB)-coupled tumor cells, to induce antitumor activity. SEB was chemically conjugated to tumor cells using a heterobifunctional cross-linking agent acting through NH2 and SH groups. V beta 8+ T cells were activated and increased in number after the culture with SEB-bound Meth A cells. The cultured T cells exhibited an antitumor activity in the Winn assay. Interleukin 2 (IL-2) receptor alpha + (IL-2R+) V beta 8+ T cells but not IL-2R+ V beta 6+ T cells increased in number in mice injected with SEB-bound Meth A cells. However, the percentages of V beta 8+ and V beta 6+ T cells did not change by this immunization. The antitumor effector cells were V beta 7- 8- CD4+ T cells. In vivo immunization with SEB-bound cells induced a strong antitumor activity, i.e., tumor-free mice/total mice = 14 of 15 (93%) for Meth A and 7 of 15 (47%) for hepatoma MH134. The induced antitumor activity was both dose dependent and tumor specific. Treatment with SEB-bound cells prolonged the survival days of Meth A-bearing mice by 62%. These results suggest that SEB-bound tumor cells may be a powerful method for induction of in vivo antitumor activity.