The steps in the heme biosynthetic pathway were evaluated in normal and in copper deficient swine. As anemia developed, the activity of heme biosynthetic enzymes increased. These data suggest that the anemia of copper deficiency is not the result of defective heme biosynthesis and, therefore, that copper is not a cofactor in any of the reactions studied. Since the morphologic characteristics of the anemia of copper deficiency suggest defective hemoglobin synthesis, it is concluded that copper is essential either for the normal metabolism of iron or for the synthesis of globin.