Measurements of iron absorption and incorporation into RBCs were obtained with the stable isotope 58Fe, administered as a reference dose, in 11 premature infants with birth weights between 780 and 1,520 g and gestational ages between 24 and 33 weeks. Each study included a timed stool and urine collection, nasogastric tube administration of a single dose of about 228 micrograms of 58Fe/kg of body weight (as FeSO4, with 10 mg/kg of vitamin C) between feedings, and blood samples before 58Fe (day 1) and then 2 weeks (day 15) later. Gastrointestinal absorption of the 58Fe dose as measured by fecal isotope balance was 41.6 +/- 17.6% (mean +/- SD). However, only 12.0 +/- 9.6% of the 58Fe dose (28.7 +/- 22.3% of the absorbed 58Fe dose) was incorporated into RBCs on day 15. 58Fe absorption and 58Fe incorporation into RBCs on day 15 were significantly correlated with the hemoglobin concentration and reticulocyte count on day 1. Transfusion history did not affect 58Fe absorption or 58Fe incorporation into RBCs. We conclude that concurrent measurement of 58Fe absorption with fecal monitoring and of 58Fe incorporation into RBCs permits a better understanding of the fate of iron ingested by premature infants than either measurement alone.