The neuropeptide Y (NPY) Y2 receptor (Y2R) is a G-protein-coupled receptor that is involved in the regulation of various physiological processes such as neurotransmitter release, bone metabolism, and memory. Consequently, the Y2R represents a potential drug target, e.g., for the treatment of epilepsy and mood disorders. Until now, the determination of the Y2R binding affinities of Y2R ligands has primarily been performed using 125I-labeled derivatives of the endogenous Y2R agonists NPY and peptide YY (PYY). A tritium-labeled NPY derivative has also been used; however, its suitability for binding assays in sodium-containing buffer is doubtful. We synthesized a tritium-labeled PYY derivative by [3H]propionylation at Lys4 ([3H]2). The radioligand was characterized by saturation binding, association, and dissociation kinetics and was applied in competition binding assays. Specific binding of [3H]2 at intact Chinese hamster ovary cells expressing the hY2R was saturable in both sodium-free buffer (apparent Kd = 0.016–0.067 nM) and sodium-containing buffer (175 mM Na+, apparent Kd = 0.16–0.18 nM). Competition binding experiments with Y2R reference ligands yielded Ki values, which are in good agreement with the reported Y2R binding affinities, showing that [3H]2 represents a useful tritiated tool compound for the determination of Y2R binding affinities also in buffers containing sodium at physiological concentrations.