We measured red blood cell iron incorporation (RBC-inc) in 13 human milk-fed premature infants (birthweight 1037 +/- 289 g, gestational age 27 +/- 2 wk, weight at start of study 1571 +/- 426 g) who were receiving full tube-feedings of human milk fortified with a commercial human milk fortifier (FortHM). The relative RBC-inc of supplemental iron (2 mg/kg/d of ferrous sulfate) was assessed using 57Fe sulfate mixed directly into a 24-h volume of FortHM, and 54Fe sulfate given as a bolus between two FortHM feedings the next day. RBC-inc was similar between the two methods of supplemental iron administration (4.7 +/- 2.5% vs 4.6 +/- 1.5%, respectively). Although these values are lower than RBC-inc expected from iron native to human milk, the relatively large amount of iron in the supplements contributed most of the iron incorporated into RBC by the infants. There was a significant positive correlation between the reticulocyte count and RBC-inc. As the high nutrient (especially calcium) content of the FortHM did not interfere with iron utilization, adding iron directly to FortHM, or incorporating it into commercial fortifiers, may be a practical method to provide iron to premature infants.