Diazepam binding inhibitor (DBI), a brain neuropeptide putative ligand for benzodiazepine binding sites, has been isolated and purified to homogeneity. This compound, like the anxiogenic beta-carbolines, injected intracerebroventricularly facilitates shock-induced suppression of drinking in thirsty rats. Cyanogen bromide (CNBr) cleavage of DBI produces three peptide fragments: the carboxy terminal fragment (F3 approximately equal to 1800 mol.wt.) and an intermediate fragment (F2 approximately equal to 3200 mol.wt.) are inactive, whereas the fragment that contains the amino terminus (F1 approximately equal to 6500 mol. wt.) facilitates punishment inhibition of operant behavior in rat. These data suggest that the F1 peptide contains the active sequence. The latter might be the natural effector of benzodiazepine recognition sites while DBI could be a polyprotein functioning as the precursor of the putative endogenous ligand of the benzodiazepine recognition site.