The spleen plays an important role in the response of the recipient's immune system to a primarily vascularized graft and cyclosporine treatment is known to alter this response. To investigate the interaction between the splenic immune response and CsA's immunosuppressive actions more thoroughly, Lewis recipients of Brown-Norway heterotopic heart grafts were treated i.p. daily with normal saline or with CsA doses of 0.75, 1.5, or 3.0 mg/kg/day from day 1 through day 50 or until rejection. Rats treated with 3 mg/kg were splenectomized intraoperatively (i.o.) or not splenectomized. Rats in subgroups of the other treatment groups were splenectomized i.o., on day 5, not splenectomized, or the recipient's spleen cells were reinfused after i.o. splenectomy. In non-CsA-treated rats, i.o. splenectomy (median survival time, [MST] = 11 days) and day 5 splenectomy (MST = 11 days) prolonged graft survival minimally in comparison with nonsplenectomized animals (MST = 7 days). Reinfusion of the spleen cells reversed this effect (MST = 7 days). Most interestingly, the immunosuppressive efficacy of 1.5 mg/kg of CsA (MST = 91 days) was reduced by day 5 splenectomy (MST = 24 days) and completely abolished by i.o. splenectomy (MST = 11 days). Spleen cell reinfusion partially restored the effect of CsA treatment (MST = 88 days). Since splenectomy resulted in a complete abrogation of the immunosuppressive efficacy of 1.5 mg/kg CsA, our results support the hypothesis that certain spleen cells augment immunosuppression by CsA. These findings provide additional evidence that the immune system's own regulation of its antigraft response can be an important component of the overall suppression of rejection that is associated with the use of certain immunosuppressive drugs.