Cytotoxic T lymphocyte–associated antigen 4 (CTLA-4) is an essential negative regulator of T cell activation. Recent evidence suggests that CTLA-4 association with the immunological synapse during contact with antigen-presenting cells is important for its inhibitory function. In the present study, we observed a direct interaction of CTLA-4 with the phosphorylated form of T cell receptor (TCR)ζ within the glycolipid-enriched microdomains associated with the T cell signaling complex. In this setting, CTLA-4 regulated the accumulation/retention of TCRζ in the signaling complex, as the lipid raft fractions from CTLA-4KO T cells contained significantly higher amounts of the TCR components when compared with wild-type littermates. In contrast, coligation of CTLA-4 with the TCR during T cell activation selectively decreased the amount of TCRζ that accumulated in the rafts. These results suggest that CTLA-4 functions to regulate T cell signaling by controlling TCR accumulation and/or retention within this a critical component of the immunological synapse.