The events that generate T cell-mediated immune tolerance in early pregnancy are ill-defined. To investigate the significance of seminal fluid Ags in activating maternal T cells, and define the underlying Ag presentation pathways, OVA-specific T cells were adoptively transferred to female mice inseminated by males ubiquitously expressing membrane-bound OVA. OVA-reactive CD8+ OT-I and CD4+ OT-II T cells transferred to mated recipients expressed activation markers CD25 and CD69 and proliferated vigorously in the para-aortic lymph nodes, but not in distal lymph nodes or spleen, and OT-I T cells expressed IFN-γ and IL-2. In contrast, OT-I T cells transferred later in pregnancy or up to 10 days postpartum expressed CD25 and CD69 and proliferated in all peripheral lymphoid tissues examined. OVA Ag was present predominantly in the plasma fraction of seminal fluid, and seminal plasma, but not sperm, was necessary for T cell proliferation. Female H-2K b bone marrow-derived cells expressing TAP were essential for OT-I T cell proliferation, but responses were not elicited by OVA Ag presented by paternal MHC in seminal fluid or associated with placental cells. This study shows that at conception, seminal fluid drives activation and expansion of paternal Ag-reactive CD4+ and CD8+ T cell populations, and female APCs have an essential role in cross-presenting Ag to CD8+ T cells via a TAP-dependent pathway. Delivery of paternal Ags and immune-deviating cytokines by seminal fluid at conception may activate Ag-dependent CD4+ and CD8+ regulatory T cells mediating tolerance of pregnancy.