Phenobarbital-N-glucoside (PNG) has recently been identified as a significant metabolite of phenobarbital (PB). Five neonates treated with PB alone for seizures were studied. Serum concentration of PB ranged from 30 to 80 mg/l. Serial single daily voided specimens were analyzed for PB, PNG, and total p-hydroxyphenobarbital (PHPB). PNG was detected on the 14th day of life in 1 patient, who was the oldest by gestational age. On the 20th day of life, PNG accounted for 50% of the dose excreted in the urine. No PNG was detected in other patients by the 12th, 15th, or 16th day of life. PHPB was found on day 4, in the first urine examined, in the patient who made PNG. In other patients, PHPB was first excreted on the 4th day of life in 2 patients and on the 10th day in 2. It appears that N-glucosidation, when activated, rapidly becomes a significant route for PB metabolism in the neonate.