The rat glucocorticoid-induced receptor (rGIR) is an orphan G protein-coupled receptor awaiting pharmacological characterization. Among known receptors, rGIR exhibits highest sequence similarity to the neuropeptide Y (NPY)–Y2 receptor (38–40%). The pharmacological profile of rGIR was investigated using 125I-PYY3–36, a Y2-preferring radioligand and several NPY analogs. rGIR displayed a similar displacement profile as reported for the Y2 receptor, in that the Y2-selective C terminus fragments of NPY and PYY (NPY3–36 and PYY3–36) showed high affinity binding and activation of rGIR (low nanomolar range). The rank order potency for displacement was NPY3–36 > PYY3–36 = NPY > NPY13–36 > Ac, Leu NPY24–36 > [D-Trp32]-NPY > Leu31, Pro34-NPY = hPP. NPY and Y2-selective agonists NPY3–36 and PYY3–36 led to significant activation of 35S-GTPγS binding to rGIR transfected cells. BIIE0246, a specific Y2 antagonist, displaced 125I-PYY3–36 binding to rGIR with high affinity (95 nM). Activation of 35S-GTPγS binding by Y2-selective agonist in rGIR transfected cells was also completely abolished by BIIE0246. Our data report, for the first time, an interaction of NPY ligands with rGIR expressed in vitro, and indicate similarities between GIR and the NPY–Y2 receptor.