The chemokine receptors CXCR3 and CXCR4 are primarily involved in memory Th1 cell-driven autoimmune diseases. Although recent studies in chronic inflammatory disease showed therapeutic success using combined blockade, details of CXCR3 and CXCR4 synergism are not understood. In this investigation, we intended to unravel the interaction of these chemokine receptors in static and dynamic cell-migration assays at both the cellular and molecular levels. Effects of combined stimulation by murine CXCL9 and CXCL12, ligands of CXCR3 and CXCR4, respectively, were analyzed using a murine central memory Th1 cell clone. Costimulation with CXCL9 desensitized the chemotaxis of Th1 cells toward CXCL12 by up to 54%. This effect was found in murine EL-4 cells, as well as in primary human T cells. Furthermore, under dynamic flow conditions CXCL12-induced crawling and endothelial transmigration of Th1 cells was desensitized by CXCL9. Subsequent experiments uncovered several molecular mechanisms underlying the heterologous cross-regulation of CXCR4 signaling by the CXCR3 ligand. CXCR4 surface expression was reduced, whereas CXCL12-induced Akt phosphorylation and intracellular Ca(2+) signals were modulated. Moreover, blockade of Rac by NSC23766 revealed differential effects on CXCL12 and CXCL9 chemotaxis and abolished the desensitizing effect of CXCL9. The desensitization of CXCR4 via CXCR3 in memory Th1 cells suggests that their in vivo homeostasis, widely regulated by CXCL12, seemed to be significantly altered by CXCR3 ligands. Our data provide a more detailed understanding for the continuing extravasation and recruitment of Th1 lymphocytes into sites of persistent inflammation.