Engagement of the Ag receptor on naive CD8+ T cells by specific peptide-MHC complex triggers their activation/expansion/differentiation into effector CTL. The frequency of Ag-specific CD8+ T cells can normally be determined by the binding of specific peptide-MHC tetramer complexes to TCR. In this study we demonstrate that, shortly after Ag activation, CD8+ T cells transiently lose the capacity to efficiently bind peptide-MHC tetramer complexes. This transient loss of tetramer binding, which occurs in response to naturally processed viral peptide during infection in vitro and in vivo, is associated with reduced signaling through the TCR and altered/diminished effector activity. This change in tetramer binding/effector response is likewise associated with a change in cell surface TCR organization. These and related results suggest that early during CD8+ T cell activation, there is a temporary alteration in both cell surface Ag receptor display and functional activity that is associated with a transient loss of cognate tetramer binding.