During the productive interaction of T cells with antigen-presenting cells (APCs), engaged receptors, including the T cell antigen receptors and their associated tyrosine kinases, assemble into spatially segregated supramolecular activation clusters (SMACs) at the area of cell contact. Here, we studied intracellular signaling in SMACs by three-dimensional immunofluorescence microscopic localization of CD3, CD45, talin, phosphotyrosine, Lck and phosphorylated ZAP-70 in T cell–APC conjugates. Two distinct phases of spatial-temporal activation, one before and one after SMAC formation, which were separated by a brief state of inactivation caused by CD45, were observed at the T cell–APC contact area. We propose that pre-SMAC signals are sufficient to activate cell adhesion, but not productive T cell responses, which require orchestrated signaling in SMACs.