The suppressive effect of rapamycin on T cells has been extensively studied, but its influence on the function of APC is less clear. The data in this study demonstrated that immunostimulatory activity of B10 (H2 b) dendritic cells (DC) exposed to rapamycin (rapa-DC) was markedly suppressed as evidenced by the induction of low proliferative responses and specific CTL activity in allogeneic (C3H, H2 k) T cells. Administration of rapa-DC significantly prolonged survival of B10 cardiac allografts in C3H recipients. Treatment with rapamycin did not affect DC expression of MHC class II and costimulatory molecules or IL-12 production. Rapamycin did not inhibit DC NF-κB pathway, however, IL-12 signaling through Janus kinase 2/Stat4 activation was markedly suppressed. Indeed, Stat4−/− DC similarly displayed poor allostimulatory activity. The Stat4 downstream product, IFN-γ, was also inhibited by rapamycin, but DC dysfunction could not solely be attributed to low IFN-γ production as DC deficient in IFN-γ still exhibited vigorous allostimulatory activity. Rapamycin did not affect DC IL-12R expression, but markedly suppressed IL-18Rα and β expression, which may in turn down-regulate DC IL-12 autocrine activation.