The transmembrane chemokine CX3CL1 is expressed on the endothelial surface and promotes leukocyte adhesion and transmigration by receptor interaction via its extracellular chemokine domain. Since little is known about its intracellular C-terminus, we examined the consequences of C-terminal truncation on cellular distribution, proteolytic shedding and function of murine CX3CL1. Full length murine CX3CL1 was expressed and shed by the metalloproteinase ADAM10 as described for human CX3CL1. Truncation of murine CX3CL1 led to reduced maturation and impaired trafficking to the surface. Truncation of CX3CL1 also abrogated localization to early endosomal vesicles, but increased shedding from the surface by ADAM10. Once truncated CX3CL1 was expressed on the surface, it mediated cell contact and induced leukocyte transmigration similar as full length CX3CL1. These data suggest that the C-terminus of CX3CL1 carries important determinants for cellular trafficking but not for function of the chemokine during leukocyte recruitment.