Riboflavin (B2) plays a central role in intermediary metabolism and energy release; little is known about riboflavin requirements or pathophysiological consequences of deficiency states in preterm (PT) infants. 68 infants were allocated randomly to diets including human milk or a preterm formula containing 1.8 mg riboflavin/1. 18 of 23 human milk fed infants, who had not received supplementary riboflavin before day 7, developed biochemical riboflavin deficiency (erythrocyte glutathione reductase activation coefficient >1.3) compared with a low incidence in the formula fed group (p<0.01). Dietary effects of riboflavin status out-weighed those of phototherapy. Further studies demonstrated significant photodegredation of riboflavin in breastmilk during home collection, handling and enteral infusion: controlled exposure to daylight of human milk in plastic storage bottles or infusion apparatus, resulted in 40-50% destruction over 6 h (also 70% vitamin A lost): these losses may compound deficiency. Phototherapy lighting was similarly destructive. Our preliminary work on neonatal and older rats demonstrate that experimental riboflavin deficiency decreases oxygen consumption (p<0.05) and decreases (p<0.05) iron utilization and fatty acid oxidation (the latter suggested also by early studies on PT neonates). We speculate that riboflavin deficiency in PT infants might impair energy metabolism and substrate utilization though early supplementation requires caution since light degraded riboflavin may release damaging free radicals.