Synthetic peptides related to triakontatetraneuropeptide (TTN) [17TQPTDEEMLFIYSHFKQATVGDVNTDRPGLLDLK50; diazepam binding inhibitor (DBI) 17-50], a natural brain processing product of rat DBI, were analyzed for their physicochemical and ligand-receptor interaction characteristics. The ability of TTN and TTN-related fragments to displace [3H]flumazenil (ethyl-8-fluoro-5,6-dihydro-5-methyl-6-oxo-4H-imidazol[1,5a] [1,4]-benzodiazepine-3-carboxylate) or [3H]Ro 5-4864 [7-chloro-1,3-dihydro-1-methyl-5-(p-chlorophenyl)-2H-1, 4-benzodiazepine-2-one] from their respective benzodiazepine (BZ) binding site subtypes was tested in intact cerebellar culture neurons or in homogenates of cultured astrocytes. These studies indicate that the C-terminal region of TTN, which is also present in DBI 22-50, eicosapentaneuropeptide (DBI 26-50), and octadecaneuropeptide (ODN) (DBI 33-50), but not in DBI 19-41, is essential for interaction with the BZ recognition sites. When the C-terminal lysine of ODN is blocked with an NH2 group, the ability of ODN to interact with the binding of [3H]flumazenil is lost. A comparison analysis of the binding data with the secondary structure characteristics of the peptides demonstrated that TTN (DBI 17-50) and DBI 22-50, which have hydrophobic portions and marked tendencies to produce alpha-helicity, specifically displace (apparent Ki, 5-6 microM) [3H] Ro 5-4864 from astroglial cell binding sites. Peptides (ODN, eicosapentaneuropeptide, OND-NH2) with very low tendencies to form alpha-helices and with virtually no hydrophobic structure were not able to displace Ro 5-4864 at concentrations of up to 100 microM. In contrast, ODN was a good displacer of [3H]flumazenil from intact neurons, with an apparent IC50 of 5 microM. These data suggest that the alpha-helical portion of TTN may be important for BZ receptor recognition and BZ receptor subtype discrimination.