PURPOSE. The purpose of this study was to determine the optimal conditions for prolonging corneal allograft survival by inducing anergy with the superantigen staphylococcal enterotoxin B (SEB). METHODS. A rat model of penetrating keratoplasty, whereby Fisher344 donor corneas are implanted into Lewis recipients, was used to evaluate the effects of SEB on inhibiting immune-mediated allograft rejection. To induce anergy, SEB was injected into the peribulbar space of Lewis rats. Furthermore, histopathology and immunofluorescent staining were used to examine the levels of infiltrating CD4(+) and CD8(+) T lymphocytes and NK1.1(+) lymphocytes. RESULTS. By administering SEB, at doses of 90 or 120 mug/kg 7 days before and after keratoplasty, we suppressed the episode of corneal graft rejection for a median of 12 and 30 days, respectively. In contrast, rejection was observed when 30 or 60 mug/kg of SEB was administered. After SEB injections, lymphocyte infiltration into the corneal grafts was reduced, and the expression of NK1.1(+) lymphocytes was enhanced, suggesting that anergy may be occurring. Also, there were no differences in the number of infiltrating CD4+ and CD8+ T lymphocytes cells between the control group and groups injected with 30 and 120 mug/kg SEB on postoperative days 10 and 30. CONCLUSIONS. Inducing anergy with the superantigen SEB prolonged corneal graft survival in a rat model of penetrating keratoplasty. Therefore, these results support the possibility of prolonging corneal allograft survival in a clinical setting by preventing immune-mediated rejection through the administration of the superantigen SEB.