In iron deficiency, zinc protoporphyrin (ZPP) is produced instead of heme, and the ZPP concentration in erythrocytes is increased (normal < or = 40 mumol/mol heme). We investigated the relevance of ZPP for staging iron deficiency. ZPP was determined by hematofluorometry in samples from 103 patients. Nineteen patients with iron depletion showed decreased serum ferritin (12.1 +/- 4.4 micrograms/L) with normal ZPP and hemoglobin (Hb). Twelve patients with iron-deficient erythropoiesis had decreased ferritin (10.4 +/- 2.4 micrograms/L), increased ZPP (72 +/- 9 mumol/mol heme), and normal Hb concentrations. In 72 patients with iron-deficiency anemia, ferritin was < 12 micrograms/L. In mild anemia (Hb between 100 and 120 g/L, and normal erythrocyte indices), ZPP was 100 +/- 16 mumol/mol heme. In severe anemia (Hb < 100 g/L, decreased erythrocyte indices), ZPP values were significantly higher (265 +/- 109 mumol/mol heme). We conclude that measurements of ZPP, ferritin, and Hb can reliably be combined to classify the degree of iron deficiency.