Ligation of MHC class I-recognizing receptors on NK cells dramatically modulates their secretory and cytotoxic function. This study focuses on characterizing key signaling events regulating these activities after ligation of the C-type lectin superfamily member, CD94. We isolated separate clonal populations of human NK cells in which ligation of CD94 (kp43) either triggered cell-mediated cytotoxicity (group A clones) or potently inhibited NK cell activation (group B clones). We then evaluated the proximal signaling events that regulate these alternative responses. CD94 stimulation of group A clones induced the rapid activation of intracellular protein tyrosine kinases (i.e., lck and ZAP-70), phospholipase C, and phosphatidylinositol 3-kinase. In contrast, CD94 ligation on group B clones had none of the above noted effects and instead inhibited the FcR-induced tyrosine phosphorylations of ZAP-70 and phospholipase C-gamma 2, the formation of phospho-zeta/ZAP-70 complexes, and the release of inositol phosphates. These results define distinct proximal signaling events initiated after CD94 ligation and suggest that clonotypic differences in signaling generate fundamentally different NK cell-mediated responses.