The human Haymaker gene, at 19q13.2 in the Leukocyte Receptor-Receptor-Related Complexes, encodes a 38.5-kDa non-MHC protein found on the plasma membrane of tumor cell lines that are highly susceptible to lysis by naive (unstimulated) natural killer (nNK) cells. We hypothesized that Haymaker might act as a ligand in the tumoricidal activity of nNK cells because this molecule was absent from the surface of malignant cell lines that were resistant to their cytolytic activity. We examined the capacity of this protein to act as a ligand in the tumoricidal activity of freshly isolated nNK cells and IL-2 activated natural killer (NK) cells (aNK cells) by performing blocking studies with recombinant Haymaker (r-Haymaker) and peptide-specific anti-Haymaker antibodies. These competition studies demonstrated that both r-Haymaker and anti-Haymaker antibodies inhibited the tumoricidal function of nNK but not aNK cells. We conclude that Haymaker likely plays an essential role as an activating ligand in nNK-cell-mediated cytotoxicity, whereas the tumoricidal activity of aNK cells is, for the most part, induced by other molecules.