Using an in vivo model of tumor/self peptide presentation for induction of class I-restricted skin test reactivity, we have previously shown that a minority population of CD8+ dendritic cells (DC) negatively regulates the induction of T cell reactivity by peptide-loaded CD8− DC in DBA/2 mice. However, the CD8− fraction can be primed by IL-12 to overcome inhibition by the CD8+ subset when the two types of DC are cotransferred into recipient hosts. We report here that exposure of CD8+ DC to IFN-γ greatly enhances their inhibitory activity on Ag presentation by the other subset, blocking the ability of IL-12-treated CD8− DC to overcome suppression. In contrast, IFN-γ has no direct effects on the APC function of the latter cells and does not interfere with IL-12 signaling. The negative regulatory effect triggered by IFN-γ in CD8+ DC appears to involve interference with tryptophan metabolism in vivo. Through tryptophan depletion affecting T cell responses, IFN-γ acting on CD8+ DC may thus contribute to regulation of immunity to tumor/self peptides presented by the CD8− subset.