Brief treatment of rhesus macaques with immunotoxin plus 15-deoxyspergualin has yielded exceptional numbers (54%) of stable tolerant kidney allograft recipients, surviving over 6 years without rejection or immunosuppression. An early increase in IL-10 and reduction in IFNgamma distinguished recipients that subsequently became tolerant. Furthermore, analysis suggested that this immune switch was programmed within hours of transplantation. Administering deoxyspergualin within 5 h of surgery gave a higher incidence of tolerance (76%) compared to administration >5 h before or after surgery (11%, P<0.01). Deoxyspergualin inhibits nuclear translocation of activated NF-kappaB through heat shock proteins. Lymph node biopsies from tolerant recipients showed significant reductions in cytoplasmic expression of Hsp70 and RelB and almost complete inhibition of nuclear translocation of both. The early timing effect of deoxyspergualin suggests a crucial limitation to induction of stable tolerance is activation of Hsp-dependent innate responses to damage by ischemia-reperfusion. This was supported by studies in murine kidney reperfusion injury, where deoxyspergualin given 5 h before reperfusion protected renal function and reduced levels of IL-6 and IL-12. The narrow timing window for initiating deoxyspergualin treatment suggests the innate immune system is poised to defeat allograft tolerance induction, so effective blockade of NF-kappaB-mediated innate immunity must be in place early, to enable development of a tolerogenic environment.