The interactions of rapamycin (Rapa), CsA, and donor-specific transfusion (DST) were examined in an ACI to Lewis rat heterotopic cardiac allograft model. Survival data were truncated at 175 days for the purpose of statistical comparisons. Vehicle (Veh)-treated animals rejected at a mean of 7.3 +/- 0.2 days. Treatment with a DST on the day before transplantation (d-1) resulted in a decreased survival of 5.5 +/- 0.3 days (P < 0.05). An 8-day course of low-dose CsA (d-1 to d6) in Veh-treated animals prolonged allograft survival to 10.3 +/- 0.5 days (P < 0.05). The addition of a DST d-1 to the Veh/CsA-treated animals resulted in a synergistic prolongation of survival to 45.7 +/- 12.5 days (P < 0.05). Rapa proved to be a potent immunosuppressive agent in this model, with an 8-day course of Rapa (d-1 to d6) at 0.5 mg/kg/day (R1) or 1.5 mg/kg/day (R2), resulting in mean allograft survivals of 24.2 +/- 5.0 and 67.3 +/- 20.9 days, respectively (P < 0.05 vs. Veh or Veh/CsA). Rapa demonstrated synergy with CsA by further prolonging allograft survival to 115.9 +/- 27.9 days (R1/CsA, P < 0.05 vs. Veh/CsA, R1) and 164.6 +/- 10.4 days (R2/CsA, P < 0.05 vs. Veh/CsA). The extended graft survival using a combination of Rapa and DST, compared to Rapa alone, did not prove to be statistically significant. When all three treatments were combined (Rapa/DST/CsA), no significant benefit to survival was seen over Veh/DST/CsA (45.7 +/- 12.5 days vs. R1/DST/CsA, 81.9 +/- 18.4 days; R2/DST/CsA, 98.5 +/- 21.3 days; P > 0.05). If Rapa was withheld until postoperative day 1 (Rapa, 1.5 mg/kg/day on d1 to d6/DST/CsA), enhancement of the DST/CsA effect was seen with a mean survival of 170.2 +/- 4.8 days (P < 0.05). This suggests that the timing of Rapa administration may significantly impact its interaction with DST/CsA treatment and merits further investigation.