The optimal evaluation of iron metabolism requires the administration of two isotopes of iron. However, high-precision measurement of isotopic ratios from blood samples obtained after administration of two stable isotopes of iron to human subjects has not previously been reported. Using a cation-exchange system to isolate iron from blood samples, we found that high-precision (< 0.2%) measurements of 58Fe/56Fe and 57Fe/56Fe could be performed using magnetic sector thermal ionization mass spectrometry. Clinical studies in four 1-year-old infants showed that this technique could be used to demonstrate a lower rate of iron absorption in small children given an iron supplement (57Fe) with milk compared to those given iron (58Fe and ferrous sulfate) with ascorbic acid. This technique will enable the evaluation of iron metabolism in populations in whom the use of radioactive iron tracers is not appropriate.