OBJECTIVE To test the utility of zinc protoporphyrin to heme ratio (ZnPP/H) as an indicator of iron status in premature infants and to evaluate the effect of oral iron supplements on oxidative injury. We hypothesized that iron sulfate supplementation would decrease the ZnPP/H in preterm infants. METHODS Infants eligible for this prospective study were: hospitalized, 24 to 32 weeks of gestation, 7 to 60 days old, feeding > or = 70 mL/kg/d, with a ZnPP/H > or = the mean for age. Iron dose was determined by the ZnPP/H. Iron status and oxidative injury were evaluated at study entry and completion. Concurrent control subjects met entry criteria but were not enrolled and were not treated with iron during the study interval. Statistical evaluation included repeated measures analysis of variance and Z-score conversions. RESULTS Entry ZnPP/H of iron-treated subjects (n = 16) and control subjects (n = 16) were not different. The ZnPP/H of iron-treated infants was lower at study end (P < .05) but did not change in control infants. Iron treatment (3 to 12 mg/kg/day) was not associated with changes in conventional measures of iron status nor in measures of oxidative injury. CONCLUSIONS Iron sulfate supplementation (3-12 mg/kg/d) decreases ZnPP/H, is tolerated, and is not associated with increased oxidative injury.