Neutrophils (PMNs) from patients with secondary iron overload have an increased iron and ferritin content as well as a phagocytosis defect. Several serum components might be incriminated in the cellular iron accumulation. We therefore compared the effects on the PMN phagocytosis of total serum as well as the ferritin and transferrin fractions of serum derived from patients with thalassemia major and healthy control subjects. An incubation system of PMNs was developed. PMN phagocytosis was measured before and after incubation. Total serum from patients with thalassemia induced a defect that was prevented by co-incubation with deferoxamine (DFO). Gel-filtration chromatography was performed to separate the serum fraction containing transferrin and albumin from that containing ferritin. The transferrin-albumin fraction had no effect on PMN phagocytosis. On the contrary, the ferritin fraction of normal serum was deleterious to PMN phagocytosis, and the same fraction from thalassemic serum decreased PMN phagocytosis even more. Co-incubation with DFO or catalase improved this defect. Moreover, a cellular increase in the L-type subunit of ferritin was observed after the incubation of PMNs with the ferritin-containing fraction from thalassemic serum. In conclusion, serum from patients with thalassemia is toxic to PMNs, and this toxicity is due to ferritin-associated iron.