Early hospital discharge of newborns is leading to collection of the newborn screening blood specimen during the first day of life in increasing numbers of newborns. There is concern that neonates with phenylketonuria who are tested this early may be missed. To examine this question, the authors screened specimens collected during the first 24 hours of life from 23 neonates at risk for hyperphenylalaninemia. The blood phenylalanine level in each of the 6 neonates with phenylketonuria and a seventh with mild hyperphenylalaninemia was greater than 2 mg/dL as early as 4 hours of age and 6 mg/dL or greater by 24 hours of age. A newborn screening phenylalanine cutoff level of 2 mg/dL would have identified all of these neonates within the first 24 hours of life, but a cutoff level of 4 mg/dL would have missed 2 of the 6 with phenylketonuria before 24 hours of life. Newborn screening programs should adopt a blood phenylalanine level of 2 mg/dL as the cutoff for suspicion of phenylketonuria and request for a second specimen. Breast-fed affected neonates had higher early blood phenylalanine elevations than formula-fed neonates, perhaps reflecting the higher protein (phenylalanine) content of colostrum.