Peptide:MHC II complexes derived from a fluorescent antigen were detected in vivo to identify the cells that present subcutaneously injected antigen to CD4 T cells. Skin-derived dendritic cells (DCs) that acquired the antigen while in the draining lymph nodes were the first cells to display peptide:MHC II complexes. Presentation by these cells induced CD69, IL-2 production, and maximal proliferation by the T cells. Later, DCs displaying peptide:MHC II complexes migrated from the injection site via a G protein-dependent mechanism. Presentation by these migrants sustained expression of the IL-2 receptor and promoted delayed type hypersensitivity. Therefore, presentation of peptide:MHC II complexes derived from a subcutaneous antigen occurs in two temporally distinct waves with different functional consequences.