The objectives of this study were to describe and compare retinol, alpha-tocopherol and gamma-tocopherol, fat, energy, and nitrogen concentrations between the foremilk and hindmilk fractions of 24-hour milk collected by 24 mothers of very preterm (< 28 weeks' gestation) infants and to relate milk vitamins A and E content to maternal vitamin intake. Concentrations of retinol were significantly higher in hindmilk than in foremilk (1.6-fold), as were concentrations of alpha-tocopherol (1.6-fold), gamma-tocopherol (1.5-fold), fat (1.7-fold), energy (1.3-fold), and nitrogen (1.05-fold). Retinol, alpha-tocopherol, and gamma-tocopherol were positively related (P < .05) to milk fat and energy but not to maternal intake. Estimates of vitamins A and E intakes of infants fed hindmilk with added human milk fortifier surpassed current recommended upper level of intakes. The higher fat-soluble vitamin content of hindmilk produced by mothers of very low birth weight infants needs to be considered in the design and recommendations for use of human milk fortifier.