Fractalkine (Fk) is a structurally unusual member of the chemokine family. To determine its role in vivo, we generated mice with a targeted disruption of CX₃CR1, the receptor for Fk. CX₃CR1^–/– mice were phenotypically indistinguishable from wild-type mice in a pathogen-free environment. In response to antibody-induced glomerulonephritis, CX₃CR1^–/– and CX₃CR1^+/+ mice had similar levels of proteinuria and injury. CX₃CR1^–/– and CX₃CR1^+/+ mice also developed similar levels of disease in myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis. We performed heterotopic MHC class I/II cardiac transplants from BALB/c mice into C57BL/6 mice. In the absence of cyclosporin A (CsA), there was no difference in graft survival time between CX₃CR1^–/– and CX₃CR1^+/+ recipient mice. However, in the presence of subtherapeutic levels of CsA, graft survival time was significantly increased in the CX₃CR1^–/– mice. Characterization of cells infiltrating the grafts revealed a selective reduction in natural killer cells in the CX₃CR1^–/– recipients in the absence of CsA and a reduction in macrophages, natural killer cells, and other leukocytes in the presence of CsA. We conclude that Fk plays an important role in graft rejection. The development of CX₃CR1 antagonists may allow reductions in the doses of immunosuppressive drugs used in transplantation.