Abstract: Neuropeptide Y (NPY), a 36-mer neuromodulator, binds to the receptors Y1, Y2, Y4 and Y5 with nanomolar affinity. They all belong to the rhodopsin-like G-protein coupled, seven transmembrane helix spanning receptors. In this study, Ala-substituted and centrally truncated NPY analogues were compared with respect to affinity to the Y-receptors. Furthermore, antibodies against the second (E2) and the third (E3) extracellular loop of NPY Y1-, Y2- and Y5-receptor subtypes were raised and affinity to intact cells was tested by immunofluorescence assays. Both methods were applied in order to receive subtype selective tools and to characterise ligand binding. The analogues [A13]-pNPY and [A27]-pNPY showed subtype selectivity for the Y2-receptor. Sera against the E2 loop of the Y1-receptor and against the E2 loop of the Y2-receptor were subtype selective. Two antibodies against the Y5 E2 and E3 loop recognised the Y5- and Y2-receptor subtypes. In combination, these sera are able to distinguish between the Y1-, Y2-, and Y5-receptor subtypes. The analogues and antibodies represent valuable tools to distinguish NPY receptors on membranes and intact cells.