The capability of NK lymphocytes to kill tumor cells depends on different receptors/ligands interactions. In order to identify the cellular ligands recognized by “orphan” triggering receptors, mice were immunized with NK susceptible target cells. mAbs were selected that inhibited NK cytotoxicity and recognized two different molecules of 70 and 60–65kDa. Tryptic digestion and mass spectra analysis of purified proteins identified these molecules as PVR and Nectin-2, respectively. PVR-Fc and Nectin-2-Fc chimeric molecules stained COS-7 cells expressing the DNAM-1 activating receptor and conversely, PVR and Nectin-2 CHO-K cell transfectants were stained by DNAM-1-Fc. Thus, both PVR and Nectin-2 represent specific ligands for DNAM-1. Importantly, the specific interaction between DNAM-1 (in NK cells) and PVR or Nectin-2 (in target cells) enhanced the NK-mediated lysis of tumor cells that was downregulated by mAb-mediated masking of the receptor or its ligands.