In the early phase of leishmaniasis three types of potential antigen-presenting cells, including epidermal Langerhans cells (LC), dermal dendritic cells (DC) and inflammatory DC, are localizedat the site of infection. Therefore, it has been a central question which cell type is responsible for the initiation of a protective immune response. In the early stage of an anti-Leishmania immune response, detectable Leishmania major antigen was localized in the paracortex of the draining lymph nodes (LN). Characterization of antigen-positive cells showed that L. major co-localized with DC of a CD11c+ CD8α– Langerin– phenotype. To determine the area of antigen uptake, dermis or epidermis, and to further define the type of antigen-transporting cells, L. major was inoculated subcutaneously and concurrently LC were mobilized with fluorescein isothiocyanate (FITC). After 3 days, DC carrying L. major antigenwere always FITC–, indicating a dermal and not an epidermal origin. Moreover, addition of L. major antigen to ex vivo isolated CD8α– and CD8α+ DC fromthe draining LN of L. major-infected C57BL/6 mice demonstrated that both DC subpopulations were able to stimulate antigen-specific T cell proliferation in vitro. Without addition of exogenous antigen only the CD8α– Langerin– DC were capable of stimulating antigen-specific T cell proliferation. Thus, we demonstrate that CD8α– Langerin– DC and not LC are the basis of the protective immune response to intracellular L. major parasites in vivo.