Upon productive interaction of CD4 T cells with antigen-presenting cells (APCs), receptors and intracellular proteins translocate and form spatially segregated supramolecular activation clusters (SMACs). It is not known whether SMACs are required for CD8 T cell activation. CD8 T cells, unlike CD4 T cells, can be activated by a single peptide-MHC molecule, or by purified monovalent recombinant peptide-MHC molecules. We studied, by three-dimensional digital microscopy, cell conjugates of fresh ex vivo CD8 T cells (obtained from OT-1 mice, which are transgenic for T cell antigen receptor reactive with the complex of H-2K^b and the ovalbumin octapeptide SIINFEKL) and peptide-pulsed APCs. Remarkably, even in T cell:APC conjugates that were formed in the presence of the lowest concentration of peptide that was sufficient to elicit T cell proliferation and IFN-γ production; the θ isoform of protein kinase C was clustered in a central SMAC, and lymphocyte function-associated antigen 1 and talin were clustered in the peripheral SMAC. Conjugation of T cells to APCs that were pulsed with concentrations of peptide smaller than that required to activate T cells was greatly reduced, and SMACs were not formed at all. APCs expressing mutant H-2K^b (Lys²²⁷) molecules that do not bind CD8 were unable to form stable conjugates with these T cells, even at high peptide concentrations. Thus, although CD8 and CD4 T cells may display different sensitivity to the concentration and oligomerization of surface receptors, SMACs are formed and seem to be required functionally in both cell types. However, unlike CD4 T cells, which can form SMACs without CD4, CD8 T cells from OT-1 transgenic mice depend on their coreceptor, CD8, for the proper formation of SMACs .