Formula supplemented with docosahexaenoic acid (DHA) improves retinal function of preterm infants but the optimal dose is unknown. In a randomized controlled trial we examined the effect of increasing the DHA concentration of human milk and formula on circulating fatty acids of preterm infants. Infants born <33 weeks gestation were fed high-DHA milk (1% total fat as DHA) or standard-DHA milk (0.2-0.3% DHA) until reaching their estimated due date (EDD). Milk arachidonic acid (AA) concentration was approximately 0.5% for both groups. At EDD, erythrocyte membrane phospholipid DHA was elevated in the high-DHA group compared with standard-DHA (mean+/-SD, high-DHA 6.8+/-1.2, standard-DHA 5.2+/-0.7, p<0.0005) but AA was lower (high-DHA 14.9+/-1.3, standard-DHA 16.0+/-1.2, p<0.0005). Feeding preterm infants human milk and formula with 1% DHA raises but does not saturate erythrocyte phospholipids with DHA. Milk exceeding 1% DHA may be required to increase DHA status to levels seen in term infants.