CTLA-4 (CD152), the CD28 homologue, is a costimulatory molecule with negative effects on T cell activation. In addition to its role in the termination of activation, CTLA-4 has been implicated in anergy induction and the function of regulatory cells. As an intracellular molecule, it must first relocate to the cell surface and be ligated, in order to inhibit activation. Although some studies have investigated CTLA-4 expression on CD4+ T cells, evidence is lacking regarding the kinetics of expression, and expression on T cell subpopulations. We have investigated CTLA-4 kinetics on human purified peripheral CD4+, naïve, memory, CD4+CD25–, CD4+CD25+ regulatory T cells, and T cell clones. Intracellular stores of CTLA-4 were shown to be very low in naïve T cells, whilst significant amounts were present in memory T cells and T cell clones. Cell surface CTLA-4 expression was then investigated on CD4+CD45RA+ (naïve), CD4+CD45RO+ (memory), CD4+CD25–, and CD4+CD25+ T cells. CD25 and CD45RO are both expressed by regulatory T cells. On naïve and CD4+CD25– T cells, CTLA-4 expression declined after four hours. In contrast, on memory and CD4+CD25+ T cells, high levels of expression were maintained until at least 48 hours. In addition, significant CTLA-4 expression was observed on T cell clones following anergy induction, indicating the potential involvement of CTLA-4 also in this form of tolerance.