Lactoferrin (Lf) is a major iron (Fe)-binding protein in human milk and has been proposed to facilitate Fe absorption. The potential effect of Lf on Fe absorption was investigated by measuring Fe absorption in infants fed breast milk (with its native content of Lf) and the same milk from which Lf had been removed (>97%) by treatment with heparin-Sepharose. Eight breast-fed infants (2–10 mo; mean age 5 mo) were fed 700 to 1000 g of each milk in a randomized, cross-over design with each child acting as his/her own control. The milk was labeled with 8.6 Mmol (0.5 mg) of58Fe and Fe absorption was measured by quantifying the incorporation of the isotope into red blood cells 14 d after intake using thermal ionization mass spectrometry. Fractional Fe absorption was significantly lower (p< 0.05) from breast milk than from Lf-free breast milk. The geometric mean (range) was 11.8% (3.4–37.4%) for breast milk and 19.8% (8.4–72.8%) for Lf-free breast milk. These results do not support a direct role for Lf in the enhancement of Fe absorption from human milk at this age. In addition, Fe absorption (11.8%) from human milk fed over several feeds was lower than that previously reported for single feed studies.