CS1 (CRACC, novel Ly9) is a novel member of the CD2 family expressed on natural killer (NK), T and stimulated B cells. Although the cytoplasmic domain of CS1 contains immunoreceptor tyrosine-based switch motifs (ITSM), which enables to recruite signaling lymphocyte activation molecule (SLAM)-associated protein (SAP/SH2D1A), it activates NK cells in the absence of a functional SAP. CS1 is a self ligand and homophilic interaction of CS1 regulates NK cell cytolytic activity. Here we have identified a novel splice variant of CS1 (CS1-S), which lacks ITSM. Human NK cells express mRNA for both wild-type CS1 (CS1-L) and CS1-S and their expression level remained steady upon various stimulations. To determine the function of each isoform, cDNA for CS1-L and CS1-S were transfected into the rat NK cell line RNK-16 and functionally tested using redirected cytotoxicity assays and calcium flux experiments. CS1-L was able to mediate redirected cytotoxicity of P815 target cells in the presence of monoclonal antibody against CS1 and a rise in intracellular calcium within RNK-16 cells, suggesting that CS1-L is an activating receptor, whereas CS1-S showed no effects. Interestingly, SAP associated with unstimulated CS1-L and dissociated upon pervanadate stimulation. These results indicate that CS1-L and CS1-S may differentially regulate human NK cell functions.