The relative contribution to cytotoxicity of each of the multiple NK cell activation receptors has been difficult to assess. Using Drosophila insect cells, which express ligands of human NK cell receptors, we show that target cell lysis by resting NK cells is controlled by different receptor signals for cytolytic granule polarization and degranulation. Intercellular adhesion molecule (ICAM)-1 on insect cells was sufficient to induce polarization of granules, but not degranulation, in resting NK cells. Conversely, engagement of the Fc receptor CD16 by rabbit IgG on insect cells induced degranulation without specific polarization. Lysis by resting NK cells occurred when polarization and degranulation were induced by the combined presence of ICAM-1 and IgG on insect cells. Engagement of receptor 2B4 by CD48 on insect cells induced weak polarization and no degranulation. However, coengagement of 2B4 and CD16 by their respective ligands resulted in granule polarization and cytotoxicity in the absence of leukocyte functional antigen-1-mediated adhesion to target cells. These data show that cytotoxicity by resting NK cells is controlled tightly by separate or cooperative signals from different receptors for granule polarization and degranulation.