The effects of a subtherapeutic regimen of rapamycin (RAPA) combined with CsA and donor-specific blood transfusions (DST) were investigated in the ACI to LEW cardiac allograft model. Three protocols were studied. In protocol 1, recipients received a DST (day -1), RAPA intravenously at 0.08 mg/kg/day (day -1 to +5), and CsA intramuscularly at 5 mg/kg/day (day -1 to +1). In protocol 2, the course of CsA was extended to day +5, and in protocol 3 the treatment was further modified to include a second DST on day +3. Control groups received drug treatments alone or combined with DST or third-party blood transfusions. In all protocols, RAPA, CsA, or DSTs alone had little or no effect on graft survival. RAPA-DST or CsA-DST also had no effects beyond the ones induced by the drugs alone. In the RAPA-CsA groups, graft survivals were similar to the ones obtained with CsA alone. In contrast, in each of the protocols, the RAPA-CsA-DST treatment resulted in significant prolongation of graft survival as compared with all controls (P < 0.05). The administration of a second DST (CsA-RAPA-DSTx2) provided no additional benefits over a single DST. Lymph node cells from CsA-RAPA-DST subjects were hyporesponsive in MLR against donor cells and suppressed the proliferation of normal LEW cells in an MLR coculture assay, suggesting the presence of suppressor cells. Furthermore, in the CsA-RAPA-DST group, the anti-donor antibody response was suppressed. These data demonstrate that RAPA, CsA, and DST interact positively by inducing a clear-cut and significant prolongation of allograft survival in the rat model under conditions in which the individual components of the treatment are essentially ineffectual.