Two fractions which inhibit benzodiazepine receptor binding were isolated from both rat and human urine. The method used involved alkaline methanolysis followed by chloroform extraction and silicic acid chromatography. This method precludes artifactual formation of esters of beta-carboline carboxylic acid (BCC) during the extraction procedure. One of the fractions (fraction E) behaved similarly to methyl ester of BCC on thin-layer chromatography and also had a similar fluorescent spectra. Administration of ethanol to male Sprague-Dawley rats decreased the concentration and the total excretion of both inhibitory fractions in a dose-dependent manner. In a clinical study on males with different family histories of alcoholism, ethanol decreased excretion of fraction E. The excretion was not affected by placebo, and it was similar in family history-positive and family history-negative subjects. These results suggest that ethanol affects the gamma-aminobutyric acid-benzodiazepine receptor complex by changing release, metabolism, and/or excretion of an endogenous benzodiazepine ligand.