Up to now neuropeptide Y (NPY) receptors, which belong to the large family of G-protein-coupled receptors and are involved in a broad range of physiological processes, are believed to act as monomers. Studies with the Y 1 -receptor antagonist and Y 4 -receptor agonist GR231118, which binds with a 250-fold higher affinity than its monomer, led to the first speculation that NPY receptors can form homodimers. In the present work we used the fluorescence resonance energy transfer (FRET) to study homodimerization of the hY 1 -, hY 2 -, and hY 5 -receptors in living cells. For this purpose, we generated fusion proteins of NPY receptors and green fluorescent protein or spectral variants of green fluorescent protein (cyan, yellow, and red fluorescent protein), which can be used as FRET pairs. Two different FRET techniques, fluorescence microscopy and fluorescence spectroscopy, were applied. Both techniques clearly showed that the hY 1 -, hY 2 -, and hY 5 -NPY receptor subtypes are able to form homodimers. By using transiently transfected cells, as well as a stable cell line expressing the hY 2 -GFP fusion protein, we could demonstrate that the Y-GFP fusion proteins are still functional and that dimerization varies from 26 to 44% dependent on the receptor. However, homodimerization is influenced neither by NPY nor by G␣ protein binding. G-protein-coupled receptors (GPCRs)1 represent a superfamily of proteins characterized by seven transmembrane ␣-helices that interact with a family of heterotrimeric GTP-binding proteins, referred to as G-proteins (1). GPCRs are found in a wide range of organisms, and many kind of chemical messengers act through them, for example adrenalin, angiotensin, or neuropeptide Y (NPY). Ligands for GPCRs are involved in a broad range of physiological functions, and their malfunction is responsible for many diseases (2, 3).Until recently GPCRs were thought to function as monomers. However, a growing number of evidence suggests that they may exist as homodimers and heterodimers (4 -9). The existence of homodimers has been shown for several GPCRs including ␤ 2 -adrenergic receptor (10 -12), ␦-and -opioid receptors (6, 13), metabotropic glutamate receptor 5 (14), calcium-sensing receptor (15-17), m3 muscarinic receptor (18, 19), vasopressin V2-receptor (20), somatostatin (21, 22), and dopamine receptors (23)(24)(25). Whereas homodimerization of the somatostatin receptor 5 (21), the ␦-opioid receptor (13), and the ␤ 2 -adrenergic receptor (11) are agonist-mediated, dimerization of the -opioid receptor (26) is agonistindependent.So far photoaffinity labeling (27), cross-linking studies (15, 24), Western blot analysis (14), and immunoprecipitation (17,28,29) are the most frequently applied methods for the investigation of receptor homodimerization. Because of the development of new fluorescent dyes, novel fluorescent proteins, and new instrumentation, the fluorescence resonance energy transfer (FRET) obtained a renaissance (30) and could be applied recently for the investigation of receptor dimerization...