RANTES (regulated upon activation, normal T cell expressed and secreted) is released by cytotoxic T lymphocytes (CTL), and is a potent chemoattractant factor for monocytes and T cells, also known for its ability to suppress HIV infection. At micromolar concentration, RANTES is able to activate leukocytes, and, paradoxically, to enhance HIV infection in vitro. These latter properties are dependent on its ability to self-aggregate. In order to understand further the mechanism of RANTES-induced activation, the effects of both aggregated and disaggregated RANTES on antigen-specific CD8(+) clones were studied in comparison with the effects of specific antigens and in the presence of specific inhibitors of RANTES-mediated activation. We observed large amounts of RANTES aggregated on the cell surface, which led to cell activation, including up-regulation of cell surface markers, and secretion of IFN-gamma and macrophage inflammatory protein (MIP)-1beta. Specific inhibitors of RANTES-induced activation, such as soluble glycosaminoglycans, MIP-1alpha and MIP-1beta, acted by preventing the binding of RANTES on the cell surface. These studies suggest that RANTES acted more like a mitogen than an antigen-independent activator.