Two subsets of murine splenic dendritic cells, derived from distinct precursors, can be distinguished by surface expression of CD8α homodimers. The functions of the two subsets remain controversial, although it has been suggested that the lymphoid-derived (CD8α⁺) subset induces tolerance, whereas the myeloid-derived (CD8α⁻) subset has been shown to prime naive T cells and to generate memory responses. To study their capacity to prime or tolerize naive CD4⁺ T cells in vivo, purified CD8α⁺ or CD8α⁻ dendritic cells were injected subcutaneously into normal mice. In contrast to CD8α⁻ dendritic cells, the CD8α⁺ fraction failed to traffic to the draining lymph node and did not generate responses to intravenous peptide. However, after in vitro pulsing with peptide, strong in vivo T cell responses to purified CD8α⁺ dendritic cells could be detected. Such responses may have been initiated via transfer of peptide–major histocompatibility complex complexes to migratory host CD8α⁻ dendritic cells after injection. These data suggest that correlation of T helper cell type 1 (Th1) and Th2 priming with injection of CD8α⁺ and CD8α⁻ dendritic cells, respectively, may not result from direct T cell activation by lymphoid versus myeloid dendritic cells, but rather from indirect modification of the response to immunogenic CD8α⁻ dendritic cells by CD8α⁺ dendritic cells.