The purpose of this study was to determine the interrelationships between iron stores, serum iron, hemoglobin, myoglobin, and cytochrome c under conditions of iron deficiency that did not interfere with normal growth. Rats were given diets containing from 7 to 500 mg iron per kilogram of diet during a period of 3 weeks of rapid growth between weaning at 21 days and approaching sexual maturity at 42 days. We found that the level of iron intake required for a maximum concentration of hemoglobin was similar to that which results in a maximum level of tissue cytochrome c. The severity of iron deficiency anemia was proportionally similar to the degree of depletion of muscle cytochrome c at all levels of iron intake below 25 mg/kg diet. The results indicate that even the mildest degree of nutritional iron deficiency anemia also affected tissue cytochrome c and could impair cytochrome-dependent mitochondrial function.