Why breastfed infants absorb extrinsic iron (EFe) exceptionally well is an unexplained phenomenon. Our objective was to identify effects of human milk fractions (HMF) on bioavailability of EFe. HMF were prepared by centrifugation followed by successive ultrafiltration using 10-, 3- and 1-kDa molecular weight cutoff membranes. EFe was added to HMF before and after treatment with digestive enzymes. Solubilization of EFe by HMF was characterized by scintillation counting of radioiron and by size exclusion chromatography/inductively coupled plasma mass spectrometry (SEC/ICPMS) of stable iron. Effects of HMF on EFe uptake and basolateral transfer were assessed by using confluent Caco-2 cells in bicameral chambers. Whey fractions of low molecular weight (MW) derived from 10-kDa filtrate, except the 1-kDa filtrate, were as effective as ascorbate and nitrilotriacetate in solubilizing EFe at intestinal pH. Basolateral radioiron transfer from Caco-2 cell monolayers was greater in the presence of low MW whey fractions than in the presence of ferrous ascorbate. The 3-kDa filtrate and 3-kDa retentate fractions promoted basolateral transfer of cellular radioiron taken up previously. SEC/ICPMS of the 1-kDa retentate fraction revealed a UV-absorbing peak of MW approximately 4.2 kDa that contained iron and that solubilized added ferric iron both before and after in vitro digestion with pepsin, pancreatin and bile extract. Our results suggested that a low MW component of breast milk whey enhances iron bioavailability. Because the iron solubilization activity is resistant to in vitro digestion, it is plausible that the component is active in vivo and may explain the excellent absorption of EFe by breastfed infants [corrected].