Many chemokine receptors are typically found on natural killer cells, including CX3CR1, the receptor for the chemokine fractalkine (FKN). This study explored whether interaction between CX3CR1 and FKN is relevant for NK cell functions in cytotoxicity against tumors. FKN expression was examined by polymerase chain reaction and CX3CR1 expression in NK cells was analyzed by flow cytometry. NK cell cytotoxicity was examined by 4-h 51Cr-release assay. FKN was expressed in a variety of tumor cell lines such as K562 cells, an NK-sensitive cell line. Approximately 90% of peripheral blood NK cells and almost all of the NK cell line, NK-92 cells, expressed CX3CR1. Anti-CX3CR1 antibody strongly neutralized the cytotoxicity of NK cells against K562 cells, and pretreatment of NK cells with recombinant soluble FKN improved the cytolytic function on tumor cells. This study demonstrates that an interaction between CX3CR1 on NK cells and FKN on tumor cells is involved in the natural cytotoxicity of NK cells against tumors.