In the present study, the subtype specificity and species selectivity of the nonpeptide BIBP 3226, as well as its in vitro antagonism of neuropeptide Y (NPY)-mediated second messengers have been investigated. Radiolabeled NPY is potently displaced by BIBP 3226 [(R)-N2-(diphenylacetyl)-N-[(4-hydroxyphenylmethyl]-D- arginine amide] on human Y1 receptor expressing Chinese hamster ovary-K1 cells (Ki = 0.47 +/- 0.07 nM). SK-N-MC human neuroblastoma cells (Ki = 5.1 +/- 0.5 nM) and the rat parietal cortex membranes (Ki = 6.8 +/- 0.7 nM). The interaction of BIBP 3226 with the Y1 receptor is stereoselective, because the (S)-enantiomer of the (R)-configured BIBP 3226 displays almost no affinity (Ki > 10,000 nM). In contrast, concentrations up to 10 microM BIBP 3226 do not displace [125I]NPY from the human Y2 receptor (neuroblastoma cell line SMS-KAN), the rabbit Y2 receptor (kidney) and the rat Y2 receptor (hippocampus). Functional antagonism could be shown for the human Y1 receptor: 0.1 microM BIBP 3226 antagonizes the NPY induced Ca++ mobilization (pKb = 7.5 +/- 0.17) as well as the NPY-mediated inhibition of cyclic AMP synthesis (pKb = 8.2 +/- 0.24) in SK-N-MC cells. In contrast, none of the formerly described putative antagonists PYX-2, [D-Trp32]NPY and benextramine could be characterized as high affinity Y1 receptor antagonists. The 18 amino acid NPY analog EXBP 68 Ile-Glu-Pro-Orn-Tyr-Arg-Leu-Arg-Tyr-NH2, cyclic (2,4'), (2',4')-diamide] displayed Y1-selective affinity with in vitro antagonistic properties (Ki = 0.33 +/- 0.04 nM and pKb = 8.4 +/- 0.07) in SK-N-MC cells. Therefore, BIBP 3226 is the first potent and subtype-selective nonpeptide Y1 receptor antagonist.