AbstractNatural killer (NK) cells are innate immune lymphocytes that provide critical defense against virally infected and transformed cells. NK-cell cytotoxicity requires the formation of an F-actin rich immunologic synapse (IS), as well as the polarization of perforin-containing lytic granules to the IS and secretion of their contents at the IS. It was reported previously that NK-cell cytotoxicity requires nonmuscle myosin IIA function and that granule-associated myosin IIA mediates the interaction of granules with F-actin at the IS. In the present study, we evaluate the nature of the association of myosin IIA with lytic granules. Using NK cells from patients with mutations in myosin IIA, we found that the nonhelical tailpiece is required for NK-cell cytotoxicity and for the phosphorylation of granule-associated myosin IIA. Ultra-resolution imaging techniques demonstrated that single myosin IIA molecules associate with NK-cell lytic granules via the nonhelical tailpiece. Phosphorylation of myosin IIA at residue serine 1943 (S1943) in the tailpiece is needed for this linkage. This defines a novel mechanism for myosin II function, in which myosin IIA can act as a single-molecule actin motor, claiming granules as cargo through tail-dependent phosphorylation for the execution of a pre-final step in human NK-cell cytotoxicity.