NKp46 has been identified in the human, rat, mouse, monkey, and cattle. We have generated a monoclonal antibody, WEN23, against rat NKp46. By flow cytometry, NKp46 is expressed by all natural killer (NK) cells but not by T cells, B cells, granulocytes, monocytes, dendritic cells, or macrophages. Thus, NKp46/WEN23 is the first NK cell-specific marker in the rat. In a redirected lysis assay, preincubation of the effector cells with WEN23 augmented lysis of the Fc receptor (FcR)⁺ murine tumor target cells, indicating that NKp46 is an activating NK cell receptor. Moreover, preincubation of the effector cells with WEN23 F(ab′)₂ fragments reduced killing of target cells, confirming the activating function of NKp46 and indicating that the mouse tumor target cells express a ligand for rat NKp46. Lysis of FcR⁻ mouse and human tumor target cells was reduced after incubation of effector cells with WEN23, suggesting that rat NKp46 recognizes a ligand that is conserved between primates and rodents. By Western blot and immunoprecipitation using WEN23, NKp46 is expressed as a monomer of ∼47 kDa in interleukin-2-activated NK cells. The immunoreceptor tyrosine-based activation motif bearing adaptor proteins CD3ζ and the γ chain of FcRI for IgE (FcɛRIγ) with NKp46 from lysates of NK cells, indicating that rat NKp46 activates NK cell cytotoxicity by similar pathways as CD16.