Epidermal Langerhans cells (LC) are immature dendritic cells (DC) located in close proximity to the site of inoculation of infectious Leishmania major metacyclic promastigotes by sand flies. Using LC-like DC expanded from C57BL/6 fetal skin, we characterized interactions involving several developmental stages of Leishmania and DC. We confirmed that L. major amastigotes, but not promastigotes, efficiently entered LC-like DC. Parasite internalization was associated with activation manifested by upregulation of major histocompatibility complex (MHC) class I and II surface antigens, increased expression of costimulatory molecules (CD40, CD54, CD80, and CD86), and interleukin (IL)-12 p40 release within 18 h. L. major-induced IL-12 p70 release by DC required interferon γ and prolonged (72 h) incubation. In contrast, infection of inflammatory macrophages (Mφ) with amastigotes or promastigotes did not lead to significant changes in surface antigen expression or cytokine production. These results suggest that skin Mφ and DC are infected sequentially in cutaneous leishmaniasis and that they play distinct roles in the inflammatory and immune response initiated by L. major. Mφ capture organisms near the site of inoculation early in the course of infection after establishment of cellular immunity, and kill amastigotes but probably do not actively participate in T cell priming. In contrast, skin DC are induced to express increased amounts of MHC antigens and costimulatory molecules and to release cytokines (including IL-12 p70) by exposure to L. major amastigotes that ultimately accumulate in lesional tissue, and thus very likely initiate protective T helper cell type 1 immunity.