Activation of T lymphocytes by antigen-presenting cells (APC) results in the formation of an immunological synapse. Following contact with the target cell, key signaling and adhesion molecules polarize within minutes to hours to the T cell-APC interface. Multispectral imaging flow cytometry, a new technology which combines flow cytometry with imaging, was used to visualize and quantify the recruitment of the CD3epsilon and Lck signaling molecules during the evolution of an immune synapse. Using this technology, thousands of T cell/macrophage conjugates could be analyzed for each experimental time point. Following Ca++ triggered T cell activation, the dynamics of Lck and CD3epsilon recruitment to the synapse, analyzed by two independent methods, were comparable. However, CD3epsilon exhibited longer residence times (>8 min) at the synapse than Lck.