We have developed new tools that allow detection of peptide–MHC II presentation to naïve CD4+ T cells in vivo. These tools were used to track early events in the immune response to a subcutaneously injected antigen. Following such an injection, antigen is quickly carried via lymphatic vessels into the draining lymph nodes, and down through thin collagen-based conduits that run through the T-cell-rich area. Langerhans cells and dermal dendritic cells near the conduits acquired antigen, produced peptide–MHC II complexes, and were the first cells to stimulate naïve antigen-specific CD4+ T cells. About 18 hours after antigen injection, dermal dendritic cells migrated from the injection site and arrived in the lymph nodes displaying a large number of peptide–MHC II complexes. Elimination of these cells truncated CD25 expression by antigen-specific CD4+ T cells and ablated the development of cell-mediated immunity. Injection of antigen with the adjuvant lipopolysaccharide allowed blood-derived myeloid and lymphoid dendritic cells to produce peptide–MHC II complexes. Antigen-specific, but not non-specific, B cells acquired antigen and produced peptide–MHC II complexes by 6 hours. These B cells migrated from random positions in the follicles to the border with the T-cell area, and interacted stably there with peptide–MHC II-specific CD4+ T cells by about 36 hours. These results demonstrate the antigen-specific CD4+ T cells interact with a variety of antigen-presenting cell types during the primary immune response.