Our understanding of the biology of human natural killer (NK) cells has significantly advanced in recent years upon identification of a family of NK cell-expressed genes that encode killer cell inhibitory receptors (KIR). Individual KIR can selectively bind various HLA class I allotypes and consequently transduce inhibitory signals that block NK cell lysis of ligand-bearing target cells. A distinct subset of related and linked genes express truncated versions of KIR that are otherwise highly homologous in amino acid sequence. Interestingly, these receptors appear to transmit stimulatory signals into NK cells and have been termed killer cell activating receptors (KAR). In this report, we demonstrate that recognition of HLA-Cw3 by the p50 KAR, NKAT8, can potentiate the cytotoxic response of appropriate NK cell clones. Specific cross-linking of this KAR with a monoclonal antibody resulted in intracellular calcium mobilization, protein tyrosine phosphorylation, and phosphorylation of the MAP kinases, ERK1 and ERK2. In addition, we identified a KAR-associated disulfide-linked dimer of a 13-kDa protein that was absent in the Jurkat T cell line and is predicted to participate in these activation signaling events. Upon treatment of NK cells with pervanadate, the disulfide-linked p13 and additional proteins of 25, 30, 37 and 50-95 kDa were identified as KAR-associated tyrosine phosphoproteins. Importantly, p13 was inducibly tyrosine phosphorylated upon cross-linking of NKAT8, which strongly suggests that the associated p13 provides KAR with appropriate cytoplasmic structure to couple with tyrosine kinase-mediated signaling effectors.