The anti-benzodiazepine monoclonal antibody 21-7F9 has been used for the identification and study of endogenous benzodiazepine-like molecules in the human, rat and bovine brains. A sandwich radioimmunoassay has been designed for the quantification of the membrane-bound endogenous benzodiazepine-like molecules. The localization of these molecules is not restricted to the brain tissue. They are also present in kidney, liver and spleen as well as in the neuroblastoma X glioma NG108-15 hybrid cell line. Immunoblots show benzodiazepine-like immunoreactivity in the membrane proteins of all of these tissues. The membrane-bound benzodiazepine-like molecules are resistant to limited proteolysis of the membranes. Moreover, this treatment increases the binding of the monoclonal antibody 21-7F9 to the membranes, probably by exposing sites that normally are not accessible to the antibody. Immunocytochemistry experiments show that benzodiazepine-like molecules are also present in samples of human cerebella that have been stored in paraffin since 1940, 15 years before the first chemical synthesis of benzodiazepines. The results indicate that the cerebellar benzodiazepine-like molecules recognized by the antibody are the product of biological (not chemical) synthesis. Benzodiazepine-like immunoreactivity has also been detected in NG108-15 cells that have been cultured for 3 months in serum-free medium. These results suggest that the cells could biosynthesize benzodiazepine-like molecules.