BACKGROUND: We previously described that a tolerogeneic regimen (TR) including (1) the infusion of a minced hamster heart suspension (MHH), (2) a single injection of an anti-natural killer (NK) cell serum (rabbit anti-asialo GM1 serum), and (3) a 4-week course of the B cell immunosuppressant leflunomide (20 mg/kg/ day) induced T cell-independent (T-I) B lymphocyte and NK cell tolerance for hamster xenoantigens in T-deficient athymic nude rats. In addition, the TR allowed for long-term hamster cardiac xenograft (Xg) survival when Xgs were transplanted 2 weeks (Day 0) after the initiation of the TR (started on Day - 14). The present study was undertaken to investigate some of the characteristics of this T-I xenotolerance in more detail. METHODS: To investigate the duration of the effect of the TR on the T-I xenotolerance, hamster Xgs were transplanted at various times after initiation of the TR. To investigate whether the maintenance of the T-I xenotolerance depended on the presence of the graft, tolerated Xgs were removed on Day +28, and the subsequent evolution of the T-I xenotolerance as well as of second hamster Xg was followed. In addition, the reversibility of NK cell nonresponsiveness by recombinant interleukin-2 was investigated in vitro. RESULTS: Xgs transplanted on day 0 or Day +7 showed long-term survival. However, all Xgs transplanted on Day +15, +30, and +60 were rapidly rejected. The latter rejection occurred in the absence of formation of anti-hamster immunoglobulin (Ig)M xenoreactive antibodies (xAbs) but correlated with the recovery of anti-hamster NK cell reactivity from day +14 on. Rejected Xgs showed infiltration of NK cells but absence of IgM xAbs or complement factor deposition. When tolerated first Xgs (transplanted on Day 0) were removed on Day +28, second hamster Xgs survived without treatment when transplanted 1 or 2 weeks later. However, second hamster Xgs transplanted 3 weeks after removal of the first Xgs were all rapidly rejected. Again, the latter rejection was characterized by the infiltration of the Xgs with NK cells and by the absence of anti-hamster IgM xAbs formation. Xenoreactive NK cell nonresponsiveness was not only shorter than xenoreactive B cell nonresponsiveness, but was also more fragile. This was evident from the fact that after addition of recombinant interleukin-2 in vitro, specific anti-hamster NK nonresponsiveness was easily broken. CONCLUSIONS: NK cell and T-I B cell xenotolerance can be induced in T-deficient rats. Compared with B cell xenotolerance, the maintenance of NK cell xenotolerance is much shorter, more dependent on the presence of the graft, and easily reversible in vitro.