In vitro, chemokines can both activate and induce migration of NK cells. However, little is known about how chemokines influence NK cell activity in vivo. We studied the role of CX 3 CL1 and its receptor, CX 3 CR1, in modulating NK cell activity in an established in vivo model of tumour cell clearance. Radiolabelled YAC-1 target cells intravenously injected into C57BL/6 mice rapidly localize to the lungs and are cleared by NK cells. In mice pre-treated with blocking anti-CX 3 CL1 or anti-CX 3 CR1 Ab, target cell clearance decreased by four- to fivefold (p<0.001). In vitro, we found no effect of anti-CX 3 CL1 or anti-CX 3 CR1 Ab on NK lysis of target cells. We further examined adhesion of NK cells to Py-4-1 endothelial cells. NK cell binding to activated endothelial monolayers was significantly inhibited by anti-CX 3 CR1 Ab or soluble CX 3 CL1 (p<0.001). These studies identify a critical role for CX 3 CL1 in modulating NK cell activity in vivo.