PD-1, an inhibitory receptor expressed on activated lymphocytes, regulates tolerance and autoimmunity. We tested the role of PD-1: PD-1 ligand (PD-L) interactions in cross-presentation and the generation and control of CD8+ responses against self-Ag. Ag-naive PD-1−/− OVA-specific OT-I CD8+ T cells exhibited exacerbated responses to cross-presented Ag in mice expressing soluble OVA under the control of the rat insulin promoter (RIP-ova high). Following adoptive transfer into RIP-ova high recipients, PD-1−/− OT-I T cells expanded in the pancreatic lymph node. In contrast to wild-type OT-I cells, PD-1−/− OT-I T cells secreted IFN-γ and migrated into the pancreas, ultimately causing diabetes. Loss of PD-1 affected CD8+ cells intrinsically, and did not significantly alter the responses of wild-type OT-I T cells adoptively transferred into the same RIP-ova high recipient mouse. PD-1: PD-L interactions also limited CD8+ effector cells, and PD-L1 expression on parenchymal tissues protected against effector OT-I T cell attack. Finally, we found that the loss of PD-1 on effector OT-I cells lowers the threshold for Ag recognition in peripheral tissues. These findings indicate two checkpoints where PD-1 attenuates self-reactive T cell responses: presentation of self-Ag to naive self-reactive T cells by dendritic cells in the draining lymph node and reactivation of pathogenic self-reactive T cells in the target organ.