The CX₃C chemokine family is composed of only one member, CX₃CL1, also known as fractalkine, which in mice is the sole ligand of the G protein-coupled, 7-transmembrane receptor CX₃CR1. Unlike classic small peptide chemokines, CX₃CL1 is synthesized as a membrane-anchored protein that can promote integrin-independent adhesion. Subsequent cleavage by metalloproteases, either constitutive or induced, can generate shed CX₃CL1 entities that potentially have chemoattractive activity. To study the CX₃C interface in tissues of live animals, we generated transgenic mice (CX₃CL1^cherry:CX₃CR1^gfp), which express red and green fluorescent reporter genes under the respective control of the CX₃CL1 and CX₃CR1 promoters. Furthermore, we performed a structure/function analysis to differentiate the in vivo functions of membrane-tethered versus shed CX₃CL1 moieties by comparing their respective ability to correct established defects in macrophage function and leukocyte survival in CX₃CL1-deficient mice. Specifically, expression of CX₃CL1^105Δ, an obligatory soluble CX₃CL1 isoform, reconstituted the formation of transepithelial dendrites by intestinal macrophages but did not rescue circulating Ly6C^lo CX₃CR1^hi blood monocytes in CX₃CR1^gfp/gfp mice. Instead, monocyte survival required the full-length membrane-anchored CX₃CL1, suggesting differential activities of tethered and shed CX₃CL1 entities.