In recent years an increasing number of sequences coding for new KIRs have been described. However, the limited availability of mAbs with unique KIR specificities has hindered an exhaustive assessment of their actual function, HLA-specificity, expression at the cell surface and distribution in different cell populations. In this study we report the generation of a novel mAb (ECM41) specific for KIR2DL3 molecules. By the use of cell transfectants expressing one or other KIR we show that this reagent allows discrimination of KIR2DL3 from other GL183 mAb-reactive molecules such as KIR2DL2 and KIR2DS2. Moreover we show that this novel mAb can be used to assess the surface expression and distribution of KIR2DL3 in different polyclonal NK populations and in NK cell clones. Along this line, we were able to analyze the HLA class I specificity of NK clones expressing either KIR2DL3 or KIR2DL2, two inhibitory receptors that were so far serologically undistinguishable. Finally, the combined use of GL183 and ECM41 mAbs in redirected killing assays allowed us to investigate the functional outcome of the simultaneous engagement of KIR2DL3 and KIR2DS2 in NK cell clones co-expressing KIRs that display opposite (inhibitory vs activating) function.