The family of human neuropeptide Y receptors (YRs) comprises four subtypes (Y1R, Y2R, Y4R, and Y5R) that are involved in the regulation of numerous physiological processes. Until now, Y4R binding studies have been predominantly performed in hypotonic sodium-free buffers using 125I-labeled derivatives of the endogenous YR agonists pancreatic polypeptide or peptide YY. A few tritium-labeled Y4R ligands have been reported; however, when used in buffers containing sodium at a physiological concentration, their Y4R affinities are insufficient. Based on the cyclic hexapeptide UR-AK86C, we developed a new tritium-labeled Y4R radioligand ([3H]UR-JG102, [3H]20). In sodium-free buffer, [3H]20 exhibits a very low Y4R dissociation constant (Kd 0.012 nM). In sodium-containing buffer (137 mM Na+), the Y4R affinity is lower (Kd 0.11 nM) but still considerably higher compared to previously reported tritiated Y4R ligands. Therefore, [3H]20 represents a useful tool compound for the determination of Y4R binding affinities under physiological-like conditions.