Abstract Superoxide (O2−) generated by the phagocyte reduced nicotinamide adenine dinucleotide phosphate oxidase is dependent on electron transfer by flavocytochrome b558 (flavocytochrome b), a transmembrane heterodimer that forms the redox center of the oxidase at the plasma or phagosomal membrane. The larger of its two subunits, gp91phox, is homologous to the yeast iron reductase subunit FRE1, and these two proteins share many structural and functional characteristics. Because FRE1 is required for iron uptake in yeast, we hypothesized that flavocytochrome b might serve a similar function in human phagocytes and thus provide a mechanism for the transferrin-independent iron acquisition observed in myeloid cells. To determine whether flavocytochrome b was required for iron uptake, we compared iron acquisition by polymorphonuclear neutrophils (PMNs) or Epstein-Barr virus (EBV)-transformed B lymphocytes derived from individuals with X-linked chronic granulomatous disease (CGD) with iron acquisition by normal cells. Our results indicate that all cells acquired iron to the same extent and that uptake could be significantly enhanced in the presence of the trivalent metal gallium. The gallium enhancement of iron uptake observed in PMNs or in EBV-transformed B lymphocytes derived from healthy individuals was mirrored by those derived from individuals deficient in flavocytochrome b. Furthermore, both normal and CGD-derived EBV-transformed B lymphocytes had similar iron reductase activity, suggesting that flavocytochrome b is not a biologically significant iron reductase. In contrast to previously suggested hypotheses, these results show conclusively that flavocytochrome b is not necessary for cellular iron acquisition, despite structural and functional similarities between yeast iron reductases and flavocytochrome b.