Myeloperoxidase, HeO2, and a halide (chloride, bromide, iodide) constitute a powerful microbicidal system which is active against a wide variety of microorganisms and is believed to contribute to the antimicrobial activity of neutrophils. The precise mechanism by which this system exerts its toxicity is unknown. We report here that the microbicidal activity of the myeloperoxidase-H2O-chloride system on Escherichia coli is associated with the loss of iron into the medium as measured by the release of 59Fe from prelabeled organisms. Iron loss (but not bactericidal activity) was considerably increased by the addition of EDTA or other iron chelators; it was not associated with a corresponding release of protein with 14C-amino-acids. Iron loss was observed with chloride or bromide as the halide, but not when iodide was employed in microbicidal concentrations. Microbicidal activity was detected at an earlier time period and at a lower halide concentration than was iron loss. Analogous changes were observed when cytochrome c was oxidized by the myeloperoxidase H2O2-halide system. The initial response was a shift in the Soret maximum, followed by a fall in absorbance accompanied by the loss of iron. As with the intact organism, iron loss was evident with chloride and bromide, but not with iodide as the halide. These findings suggest that microbial iron centers are a target for the myeloperoxidase-mediated antimicrobial system and that their oxidation may contribute to microbicidal activity.