Erythrocyte protoporphyrin (EP) was measured in 50 normal control subjects, 22 iron-responsive anemic subjects, and in 106 patients with thalassemic diseases. All normal subjects had EP of less than 80 micrograms/dL red blood cells, whereas all iron-deficiency subjects had EP of more than 80 micrograms/dL red blood cells. Six of 22 heterozygotes for thalassemias had elevated EP, and all of these had transferrin iron saturation of less than 16%, reflecting a complicating iron deficiency. Among 52 patients with beta-thalassemia/hemoglobin (Hb) E disease, 26.9% had elevated EP levels, and among 32 patients with Hb H disease, 40.6% had elevated EP. These elevated EP levels were associated with transferrin iron saturation between 18 and 44%. In none of the thalassemic patients with transferrin iron saturation above 44% was EP elevated. These findings suggest that elevation of EP in some thalassemic patients causally is related to iron supply inadequate for the massively expanded erythropoiesis. This relative iron deficiency in thalassemia occurs at a transferrin iron saturation level usually considered to be normal. These relationships demonstrate the need for an increased iron supply in patients with erythroid marrow hyperplasia, if erythropoiesis is to proceed at maximal rates.