The rational design of vaccines that elicit CD8+ T cell responses requires knowledge of the identity of the antigen-presenting cell (APC), the location and time of presentation and the nature of the antigen presented by the APC. Here we address these questions for an antigen encoded by a recombinant vaccinia virus. We found that, following local infection, vaccinia virus infected macrophages and dendritic cells in draining lymph nodes. However, only the dendritic cells presented antigen to naïve CD8+ T cells, as determined by direct visualization of sectioned nodes by confocal microscopy. Presentation occurred as rapidly as 6 h after inoculation and quickly declined in parallel with the number of infected cells present in the nodes. These data provide direct evidence that virus-infected APCs prime naïve CD8+ T cells in vivo.