Cyanine-5-labelled neuropeptide Y (NPY) was demonstrated to be an ideal universal fluorescent ligand for the combined investigation of NPY Y-1, Y-2 and Y-5 receptors. With respect to improved stability, detection of receptor subtypes in cells and tissues, and prevention of receptor internalization, small nonpeptidic fluorescent 1 antagonists should be superior. Here we present a set of four fluorescent nonpeptide NPY Y, receptor (Y,R) antagonists. The highest affinity was obtained by labelling an N-G-(6-aminohexanoyl)argininamide derived from the Y1R antagonist BIBP 3226, with Py-1, a small pyrylium dye. The fluorescent pyridinium-type Y,R antogonist, compound 4 had K, values of 29 nM and 2.7 nM, which were determined by radioligand binding and flow cytometry under equilibrium conditions, respectively; 4 had a K-b value of 0.6 nM (Ca2+ assay). The large Stoke's shift (547 vs. 615 nm) in buffer (PBS, pH 7.4) in the presence of 1% BSA and the red emission (quantum yield 56 %) are advantageous with respect to the signal-to-noise ratio. The new probe was successfully used in fluorescence-based binding experiments evaluated by flow cytometry and confocal microscopy; this demonstrates the potential of pyrylium dyes for the preparation of fluorescent ligands that are applicable for the study of G protein-coupled receptors on, living cells.