NK cell signal integration results from a balance of multiple signals triggered by activating and inhibitory receptors, ultimately determining NK cell functions. However, how precisely the combination of these antagonistic signals occurs, rapidly, upon immunological synapse (IS) formation, is not yet understood. To study the co-localisation of activating and inhibitory receptors upon IS formation, the co-localisation of NKG2D-GFP2 (short and long isoform) and Ly49A-RFP, on a nanometer scale, was investigated by measuring Förster resonance energy transfer (FRET) between both fluorescent-tagged receptors. The NK cell IS was studied using NKG2D-GFP- and Ly49A-RFP-expressing primary NK cells ex vivo from retrogenic mice, in cell-cell conjugates with NIH3T3 target cells, with or without expression of H2-Dd SCT and H2-Dd-CD4 SCT MHC-I molecules (a native and elongated version of the Ly49A ligand, respectively). The results demonstrate that NKG2D-GFP (both isoforms) efficiently associates with DAP10 or DAP12 adaptor proteins. Both NKG2D-GFP and Ly49A-RFP are expressed at the cell surface of primary NK cells and NKG2D-GFP accumulates in the NK IS upon contact with H60-expressing target cells. Regions of nanometer-scale FRET co-localisation between NKG2D and Ly49A were dependent on the presence of ligand on target cells and its dimensions. In the presence of both NK cell ligands, NKG2D and Ly49A co-localise upon IS formation. Interestingly, increasing the size of the Ly49A ligand was shown to decrease their co-localisation, decreasing FRET. In conclusion, a differential Ly49A and NKG2D co-localisation upon NK IS formation, dependent on ligand size,, may provide a molecular mechanism for NK cell signal integration. This study presents evidence, at a nanometer scale, suggesting that immune cell signal integration is achieved by a differential co-localisation of activating and inhibitory receptors upon synapse formation.