The newly developed, purported non-peptide neuropeptide Y Y1 receptor antagonist BIBP3226 was evaluated for its potential effect on the recently characterized Y3 receptor subtype and for its apparent affinity in rat and human brain membrane binding assays using highly selective neuropeptide Y Y1 and Y2 radioligands. BIBP3226 potently blocked (pA2 = 7.36) the contractile effect of neuropeptide Y in the rabbit saphenous vein, a Y1 receptor bioassay and demonstrated nM affinity for Y1/[125I][Leu31,Pro34]peptide YY binding sites. In contrast, it failed to antagonize the biological effects of neuropeptide Y in the rat vas deferens (Y2) and rat colon (Y3) and did not significantly competed for Y2/[125I]peptide YY-(3-36) binding sites in rat and human brain homogenates. Taken together, the results demonstrate further the high potency and selectivity of BIBP3226 for the neuropeptide Y Y1 receptor by establishing its lack of antagonist activity on the Y3 subtype.