The chemokine RANTES is a potent agonist of T cell activation. In an investigation of signal-transduction events activated by this chemokine, we have shown that RANTES stimulates dose-dependent phospholipase D (PLD) activity in Jurkat cells. Equilibrium-binding analyses using 125I-labeled RANTES indicated the presence of a receptor for RANTES on these cells, which has a Kd of 0.1 nM, is expressed at approximately 600 sites per cell, and a binding specificity that was not comparable with that of any of the known chemokine receptors, since 125I-labeled RANTES was displaced by macrophage-inflammatory protein-1 beta (but not macrophage-inflammatory protein-1 alpha), monocyte-chemotactic protein-1 (MCP-1), MCP-3, MCP-4, and eotaxin. RANTES-induced PLD activation was augmented by GTP gamma S, but not GDP beta S, and inhibited by the protein kinase C inhibitor bisindolylmaleimide, as well as the fungal metabolite brefeldin A, and C3 exoenzyme (Clostridium botulinum), implicating the activation of RhoA. RANTES also induced GTP-GDP exchange of immunoprecipitated RhoA. RANTES-stimulated PLD activity was dependent on an ADP-ribosylation factor(s), as assessed by inhibition studies using a synthetic inhibitory peptide of the N-terminal 16 amino acids of ADP-ribosylation factor 1. These studies indicate the potential existence of a novel receptor-mediated mechanism for activation of T cells by the chemokine RANTES.