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.