Previously, the N-glucosylation of phenobarbital had been observed only in humans. The results of a species screen (mouse, rat, guinea pig, rabbit, cat, dog, pig, and monkey) found that only mice excreted the N-glucosides of phenobarbital in urine after ip administration of sodium phenobarbital. The major diastereomer excreted by the mouse had the R configuration at the C-5 position of the barbiturate ring. The N-glucoside metabolites accounted for a small percentage of the dose (∼0.5%). Following ip dosing of the mouse with the phenobarbital N-glucosides, free phenobarbital could be detected in the urine. Upon ip or intercerebroventricular (icv) injection of the phenobarbital N-glucosides, minimal CNS activity was observed in the mouse.