Neuropeptide Y (NPY) is a potent inhibitor of neurotransmitter release through the Y2 receptor subtype. Specific antagonists for the Y2 receptors have not yet been described, Based on the concept of template-assembled synthetic proteins we have used a cyclic template molecule containing two p-turn mimetics for covalent attachment of four COOH-terminal fragments RQRYNH(2) (NPY 33-36), termed T-4-[NPY(33-36)](4). This structurally defined template-assembled synthetic protein has been tested for binding using SK-N-MC and LN319 cell lines that express the Y1 and Y2 receptor, respectively. T-4-[NPY(33-36)](4) binds to the Y2 receptor with high affinity (IC50 = 67.2 nM) and has poor binding to the Y1 receptor. This peptidomimetic tested on LN319 cells at concentrations up to 10 mu M shows no inhibitory effect on forskolin-stimulated cAMP levels (IC50 for NPY = 2.5 nM). Furthermore, we used confocal microscopy to examine the NPY-induced increase in intracellular calcium in single LN319 cells, Preincubation of the cells with T-4-[NPY(33-36)](4) shifted to the right the dose-response curves for intracellular mobilization of calcium induced by NPY at concentrations ranging from 0.1 nM to 10 mu m. Finally, we assessed the competitive antagonistic properties of T-4-[NPY(33-36)](4) at presynaptic peptidergic Y2 receptors modulating noradrenaline release, the compound T-4 [NPY(33-36)](4) caused a marked shift to the right of the concentration-response curve of NPY 13-36, a Y2-selective fragment, yielding a pA2 value of 8.48. Thus, to our best knowledge, T-4-[NPY(33-36)](4) represents the first potent and selective Y2 antagonist.