The effects of barbituric acid and phenobarbital upon carbohydrate metabolism in mice were compared. An intraperitoneal dose of 100 mg/kg of barbituric acid increased blood glucose concentrations during an intravenous glucose tolerance test, but did not alter the rate of glucose disappearance from the blood. Barbituric acid also antagonized the hypoglycemic effect of intravenously administered tolbutamide. The same dose of phenobarbital had no effect. An in vitro concentration of 100 mug/ml of barbituric acid decreased the responsiveness of isolated mouse pancreatic islets to glucose stimulation (3.0 mg/ml D-glucose). Again phenobarbital, 100 mug/ml, was without effect. The structural similarities between barbituric acid, tolbutamide and alloxan suggest that the effects observed in these experiments might reflect a competition for binding to reactive sites on or within the pancreatic B-cell.