The rigorous median-effect analysis was used to assess the interactions between cyclosporine and drugs that inhibit nucleotide synthesis pathways. Using in vitro proliferation assays wherein human lymphocytes were triggered by phytohemagglutin, anti-CD3 monoclonal antibody, or mixed lymphocyte reactions, CsA was shown to display additive interactions with 6-mercaptopurine (6-MP), mizorbine (MZB), and mycophenolic acid (MPA), and a synergistic interaction with brequinar (BQR). In the in vitro assays, BQR contributed a further synergistic effect to the double-drug combination CsA/rapamycin (RAPA). Of the four inhibitors of nucleotide synthesis pathways, only BQR noncompetitively inhibited IL-2-stimulated proliferation of the CTLL-2 cell line. Using the in vivo assay of heterotopic Buffalo (BUF, RT-1b) cardiac allografts in Wistar-Furth (WFu, RT-1u) hosts, oral administration of BQR displayed about 100% bioavailability--which, like the bolus intravenous (i.v.) mode, was eight-fold more effective than continuous i.v. infusions. Furthermore median-effect analysis of serial amounts of orally administered BQR demonstrated that it contributes synergistically to the immunosuppressive effects of intravenously delivered CsA/RAPA (0.5/0.01 mg/kg/day). The degree of synergism was proportionate to the extent of the immunosuppression. These findings document the potency of the CsA/RAPA/BQR triple-drug combination and suggest that the synergistic effects may permit dose reductions of each component, thereby mitigating toxicities resulting from the large amounts of individual agents necessary to achieve allo-unresponsiveness.