Human NK cells can be activated by a variety of different cell surface receptors. Members of the SLAM-related receptors (SRR) are important modulators of NK cell activity. One interesting feature of the SRR is their homophilic interaction, combining receptor and ligand in the same molecule. Therefore, SRR cannot only function as activating NK cell receptors, but also as activating NK cell ligands. 2B4 (CD244) is the only SRR that does not show homophilic interaction. Instead, 2B4 is activated by binding to CD48, a GPI-anchored surface molecule that is widely expressed in the hemopoietic system. In this study, we show that 2B4 also can function as an activating NK cell ligand. 2B4-expressing target cells can efficiently stimulate NK cell cytotoxicity and IFN-gamma production. Using soluble receptor fusion proteins and SRR-transfected cells, we show that 2B4 does not bind to any other SRR expressed on NK cells, but only interacts with CD48. Lysis of 2B4-expressing target cells can be blocked by anti-CD48 Abs and triggering of CD48 in a redirected lysis assay can stimulate NK cell cytotoxicity. This demonstrates that 2B4 can stimulate NK cell cytotoxicity and cytokine production by interacting with NK cell expressed CD48 and adds CD48 to the growing number of activating NK cell receptors.