C57BL/6 (B6) mice were injected i.v. with class I H-2-disparate B10.QBR spleen cells (10(7)/mouse). This regimen, termed donor alloantigen-specific i.v. pre-sensitization (DSP), induced almost complete reduction of the anti-B10.QBR mixed lymphocyte reaction (MLR) that has been regarded to represent a cytotoxic T lymphocyte (CTL)-independent graft rejection pathway. Because the DSP regimen failed to affect the generation of CTL responses, it did not prolong graft survival. Repeated (four or 11 times) administration in vivo (during 5 or 18 days) of rapamycin at suboptimal doses (0.5-2.0 mg/kg/day) failed to eliminate the capacities to exhibit MLR as well as to generate CTL responses. The suppression of CTL responses was achieved only through the combination of these two treatments. It was also shown that prolongation of skin graft survival was not induced by either of a single DSP or rapamycin treatment alone, but by the combination of these. Potent suppression of CTL responses was also induced when a single DSP was combined with repeated injection of a suboptimal dose (0.75 mg/kg/day) of another immunosuppressive drug, FK506, instead of rapamycin. However, there was a substantial difference in cellular mechanisms underlying the suppression of CTL responses by the above two different combinations. Under conditions in which lymphoid cells from mice receiving the treatments with DSP plus FK506 failed to generate CTL responses, the addition of recombinant IL-2 (rIL-2) to cultures restored the CTL generation, suggesting that CTL precursors themselves are not attenuated by the combined treatment.(ABSTRACT TRUNCATED AT 250 WORDS)