Endozepines represent a novel family of regulatory peptides that have been isolated by their ability to displace benzodiazepines from their binding sites. All endozepines derive from an 86 amino acid precursor polypeptide called diazepam binding inhibitor (DBI), which generates, through proteolytic cleavage, several biologically active endozepines. The aim of the present study was to compare the molecular forms of endozepines present in different regions of the rat brain and in various peripheral organs using an antiserum raised against the central (biologically active) region of DBI. Combination of HPLC analysis and RIA detection revealed the existence of two major forms (peaks I and II) of endozepine-immunoreactive peptides. The retention times of the two peaks (36 and 39 min, respectively) were identical in all tissues or organs tested. Western blotting analysis of cerebral cortex extracts confirmed the existence of two immunoreactive species with apparent molecular weights 4000 and 6000 Da, which respectively correspond to peaks I and II. Tryptic digestion of peaks I and II generated a single immunoreactive peptide that coeluted with the synthetic octadecaneuropeptide ODN [DBI(33-50)]. These results show that, in different parts of the brain and in various peripheral organs, DBI is rapidly processed to generate two peptides of apparent molecular weight of 4000 and 6000 Da, which both possess the biologically active determinant of endozepines.