T lymphocyte receptor CTLA-4 binds costimulatory molecules CD80 (B7-1) and CD86 (B7-2) with high avidity and negatively regulates T cell activation. CTLA-4 functions at the cell surface, yet is primarily localized in intracellular vesicles. Here, we demonstrate cycling of CTLA-4 between intracellular stores and the cell surface. Intracellular vesicles containing CTLA-4 overlapped with endocytic compartment(s) and with perforin-containing secretory granules. Cell surface expression of CTLA-4 was rapidly increased by raising intracellular calcium levels. During T cell activation, intracellular and cell surface CTLA-4 became focused towards sites of TCR activation. Cycling and directional control of CTLA-4 expression may regulate its functional interaction with APCs bearing peptide-MHC complexes of appropriate specificity and avidity.