Members of the murine Ly49 family of receptors have been shown to inhibit and activate NK cell function. Subsets of Ly49-expressing NK cells mediate the rejection of bone marrow cell allografts and the lysis of allogeneic lymphoblasts. In this report we have studied Ly49-mediated positive and negative signaling in an in vitro cytotoxicity assay using sorted NK cell subsets as effectors and a panel of 51Cr-labeled Con A lymphoblasts as targets in the presence or the absence of Abs to Ly49 and/or class I molecules. Our results demonstrate that the activating receptor Ly49D delivers stimulatory signals for target cell lysis upon interacting with H2-Dd, Dr, and Dsp2, but not H2b or H2k class I Ags. On the other hand, the inhibitory receptor Ly49G2 delivers negative signals for target cell lysis upon interacting with Dd, Dr, and H2k, but not H2b or Dsp2, class I Ags. Furthermore, Ly49-mediated negative signaling dominates Ly49D-mediated positive signaling. Thus, lysis of class I MHC-bearing targets by NK cells is not merely the consequence of the absence of an Ly49-mediated negative signal, but also requires positive recognition of class I molecules by certain Ly49 receptors. Activation of NK cells by nonself class I molecules was not predicted by the missing self hypothesis.