Brucella abortus are Gram negative facultative intracellular bacteria, which survive and replicate in host macrophages. We have recently demonstrated that activation of macrophages with interferon-gamma increases their anti-brucella activities but does not result in elimination of intracellular brucellae. Here we demonstrate that iron-loaded macrophages have an enhanced capacity to kill or prevent replication of intracellular brucellae. Iron added bound to transferrin or as a salt, iron-nitrilotriacetate, can mediate the effect. Macrophages supplemented with iron-loaded transferrin in addition to activation with interferon-gamma can frequently eliminate the intracellular organisms by 48 hr after infection. The effect is apparent following phagocytosis of either nonopsonized or antibody-opsonized brucellae, and with both attenuated and virulent strains of B. abortus. The killing can be blocked by the hydroxyl radical scavengers mannitol and thiourea. This is consistent with the Haber-Weiss reaction, in which iron catalyzes the generation of hydroxyl radicals from hydrogen peroxide.