As a basis for understanding the mechanism of action of barbiturates, a study of their inhibitory properties on anaerobic glycolysis was undertaken. In starved rats received single intraperitoneal injections of 50 mg/kg pentobarbital or of 100 mg/kg phenobarbital, there was observed a significant decrease in the blood levels of pyruvate and lactate at the time of maximal depression of motor activity. In the brain, the levels of phosphorylated metabolites of glucose were slightly decreased. Anaerobic glycolysis in vitro was found to be inhibited at the concentrations of drugs tested. The activities of hexokinase, phosphofructokinase and pyruvate kinase were significantly inhibited in the presence of the drugs. Aldolase, glyceraldehyde-3-phosphate dehydrogenase and lactate dehydrogenase were insensitive to the drugs. These findings support that barbiturate suppresses anaerobic glycolysis through inhibition of activities of enzymes which catalyze irreversible steps of glycolysis.