Mothers of VLBW infants fractionated their milk into foremilk (first 3 minutes of pumping) and hindmilk (remainder of milk collected) over 24h on postpartum day 24 (21--30). Study objectives were to: (a) compare concentrations of vitamins A and E, fatty acids, energy and nitrogen between fractions, (b) relate milk vitamins to milk fat and energy concentrations and to maternal vitamin intake. Hindmilk contained significantly higher concentrations of retinol (50%), alpha-tocopherol (80%), gamma-tocopherol (70%), total fatty acids (70%), energy (30%) and nitrogen (3%) than foremilk. Concentrations of milk retinol, alpha-tocopherol, and gamma-tocopherol were positively associated (p < 0.05) with milk total fatty acids and energy concentrations. Fatty acid profile of hindmilk differed slightly but significantly from foremilk. Maternal vitamin intake was not related to milk vitamin content. Preterm hindmilk is more energy dense than foremilk due to higher fat content, and contains increased concentrations of fat-soluble vitamins A and E.