Phototherapy with blue light decomposes riboflavin, which has a maximum absorption at 450 nm. A study was designed to determine whether riboflavin deficiency developed in neonates who received phototherapy for moderate hyperbilirubinemia. Twenty-one infants with normal erythrocyte glucose-6-phosphate dehydrogenase activity were investigated. Five infants with moderate hyperbilirubinemia who did not require phototherapy served as the controls. Riboflavin deficiency was determined from the degree of saturation of erythrocyte glutathione reductase, a method shown to reflect riboflavin nutritional status in the neonate. Sixteen of 21 infants who were exposed to phototherapy developed riboflavin deficiency; all who had phototherapy for 49 hours or more developed the deficiency. That the concentration of serum bilirubin or the duration of hyperbilirubinemia was not a factor is supported by the fact that none of the controls became deficient. This observation may have important metabolic and clinical consequences for the neonate.