CD28 and CTLA-4 are opposing regulators of T cell activation, triggered by the two ligands CD80 and CD86. How these ligands promote either T cell activation via CD28 or inhibition via CTLA-4 is not understood. Using CD80 and CD86 molecules expressed on transfected cells, we have identified a major difference between these ligands in that CD80 transfectants have the ability to inhibit activation of resting human peripheral blood T cells via interaction with CTLA-4, whereas CD86 transfectants do not. Rather, CTLA-4-CD86 interactions appear to contribute towards T cell proliferation. We also observed that CTLA-4 function is strongly influenced by TCR stimulation, effects being observed only at relatively low levels of TCR stimulation. The kinetics of CD80-CTLA-4 interactions revealed that CTLA-4 inhibition took place within the first 8 h of T cell stimulation, despite there being little measurable CTLA-4 expression on the majority T cells. However, significant amounts of CTLA-4 were observed in the CD25(+) CD4(+) subset of T cells which, when removed from the cultures, accounted for the CTLA-4 inhibition observed. Overall, these data provide evidence that CD80 and CD86 differ in their interactions with CTLA-4 and that CD80 appears to be the preferential inhibitory ligand for CTLA-4 working via a population of CD4(+) CD25(+) CTLA-4(+) regulatory T cells.