Immune responses at mucosal sites are thought to be initiated in the draining lymph nodes, where dendritic cells present viral antigens and induce naive T cells to proliferate and to become effectors. Formal proof that antigen-presenting cells (APC) do indeed localize to the regional lymph nodes has been lacking for viral infections of the respiratory tract. Influenza virus was detected in the draining mediastinal lymph nodes (MLN) early after intranasal inoculation, with peak virus titers in this tissue measured at 2 days postinfection. Virus-specific cytotoxic T-lymphocyte responses were first detected in the MLN 1 day later. Macrophages, dendritic cells, and B lymphocytes were isolated from influenza virus-infected mice and assayed for the capacity to stimulate a major histocompatibility complex class I-restricted virus-specific T-cell hybridoma. All APC populations from lungs and MLN contained virus and thus had the potential to present antigen to CD8+ T cells. The APC recovered from the lungs of influenza virus-infected mice and dendritic cells from the MLN were able to stimulate virus-specific responses. The lack of a virus-specific T-cell response to B cells corresponds to the small number of virus-positive B lymphocytes in the MLN. These results indicate that dendritic cells and macrophages are antigen positive in mice acutely infected with an influenza A virus and that dendritic cells are probably responsible for initiating the cytotoxic T-lymphocyte response to influenza virus in the draining lymph nodes.