T cells interacting with antigen presenting cells (APCs) form polarized adhesion zones between the cells. T cell surface molecules involved in both adhesion and signaling, including the T cell antigen receptor/CD3 complex (CD3/Ti) and cytoskeletal elements, redistribute to the region of contact. However, little is known about the structure and composition of the membrane at this site. Using immobilized anti-CD3 antibodies to mimic T cell-APC interactions and stimulate T cells, we have characterized the site of T cell attachment. Two sets of studies were carried out. First, we found that several anti-CD3 antibodies induced characteristic changes in attachment site shape, microfilament morphology, and the apparent molecular weight of p56{dollar}\sp{lcub}lck{rcub}{dollar}, a protein tyrosine kinase (PTK), during the first 30 minutes of activation. After 12-96 hours, anti-CD3 stimulated cells exhibited growth arrest. These changes were not elicited by immobilized anti-CD7, -CD11a or -CD71 antibodies, and were inhibited by the microfilament disrupting agent, cytochalasin D. Second, the attachment site was isolated using the detergent saponin, and shearing. The substrate attached material (SAM) represented 4.8 {dollar}\pm{dollar} 0.8% of the total cell protein and consisted of a patch of membrane, oriented with its cytoplasmic surface exposed to the medium. Biochemical analysis of SAM revealed the CD3/Ti complex only when stimulatory anti-CD3 (CD3-SAM) antibodies were used. This association was dependent on time, temperature, and antibody specificity, arguing for its selectivity. CD3-SAM also showed rapid and sustained enrichment in p56{dollar}\sp{lcub}lck{rcub}{dollar} and several phosphotyrosinylated proteins. Non-stimulatory antibodies failed to induce localization of these molecules to the SAM. Stimulation of cells with PMA and ionomycin prior to attachment with non-stimulatory antibodies did induce the localization of p56{dollar}\sp{lcub}lck{rcub}{dollar}, but not of any other PTK substrates or the CD3/Ti complex, to the SAM. These are the first studies showing the selective association of tyrosine phosphorylated proteins to a region of the membrane associated with CD3/Ti mediated signaling. Thus, T cell activation involves the active reorganization of the cytoskeleton, the CD3/Ti complex and specific tyrosine phosphorylated proteins to the site of signaling.