NK lytic-associated molecule (NKLAM) is a protein involved in the cytolytic function of NK cells. It is weakly expressed in resting NK cells but upon target cell stimulation or after incubation with cytokines that enhance NK killing, NKLAM mRNA levels increase and protein is synthesized and is targeted to cytoplasmic granule membranes. We have previously shown that NKLAM plays a role in perforin/granzyme-mediated cytolysis in vitro. To further investigate the function of NKLAM in NK cell-mediated cytotoxicity, we generated, by gene targeting, NKLAM-deficient mice. These mice have normal numbers of NK cells and other lymphoid populations in the spleen. They also have no alterations in NK maturation or NK receptor repertoire. NK cells from NKLAM-deficient and WT mice have comparable amounts of perforin, granzyme B, and lysosomal membrane-associated protein 1 (CD107a) in their cytotoxic granules and comparable levels of granule exocytosis are induced by PMA and calcium ionophore A23187. However, NKLAM-deficient NK cells display significantly less NK cytotoxic activity in vitro than WT NK cells. They also secrete less IFN-gamma upon target cell stimulation, In addition, NKLAM-deficient mice exhibit greater numbers of pulmonary metastases after i.v. injection with B16 melanoma cells. These studies indicate that NKLAM-deficient mice have diminished capacity to control tumor metastases and support the role for NKLAM in NK function both in vitro and in vivo.