Antigen-specific CD8⁺ T cells are required for the clearance of most viral infections and several cancers. However, it is not clear in vivo whether CD8⁺ T cells can engage multiple targets simultaneously, engagement results in the formation of an immunologic synapse or molecules involved in CD8 function are redistributed to the synapse. We used here high-resolution microscopy to visualize interactions between virus-specific effectors and target cells in vivo. Using either in situ tetramer staining or green fluorescent protein–labeled virus-specific T cells, we have shown that a single CD8⁺ T cell can engage two or three targets, a synapse occurs at the site of engagement and molecules involved in attachment (lymphocyte function–associated antigen 1), signaling (Lck) and lytic activity (perforin) are differentially positioned on the T cell. In addition, we have established an in vivo approach for assessing the intricacies of antigen-specific T cell activation, migration, engagement, memory and other defining elements of adaptive immunity.