BACKGROUND: Iron supplements are often recommended for older breast-fed infants, but little is known about factors affecting iron absorption from human milk or supplements. OBJECTIVE: We investigated the effects of age, iron status, and iron intake on iron absorption in healthy, term, breast-fed infants. DESIGN: Twenty-five infants were randomly assigned to receive either 1) iron supplements (1 mg x kg(-1) x d(-1)) from 4 to 9 mo of age, 2) placebo from 4 to 6 mo and iron supplements from 6 to 9 mo, or 3) placebo from 4 to 9 mo. Infants were exclusively breast-fed to 6 mo and partially breast-fed to 9 mo of age. Iron absorption was assessed by giving (58)Fe with mother's milk at 6 and 9 mo. Blood samples were obtained at 4, 6, and 9 mo, and complementary food intake was recorded at 9 mo. RESULTS: At 6 mo, mean (+/-SD) fractional iron absorption from human milk was relatively low (16.4 +/- 11.4%), with no significant difference between iron-supplemented and unsupplemented infants. At 9 mo, iron absorption from human milk remained low in iron-supplemented infants (16.9 +/- 9.3%) but was higher (P = 0.01) in unsupplemented infants (36.7 +/- 18.9%). Unexpectedly, iron absorption at 9 mo was not correlated with iron status but was significantly correlated with intake of dietary iron, including supplemental iron. CONCLUSIONS: Changes in the regulation of iron absorption between 6 and 9 mo enhance the infant's ability to adapt to a low-iron diet and provide a mechanism by which some, but not all, infants avoid iron deficiency despite low iron intakes in late infancy.