We tested the hypothesis that oxidative stress and biological effect after ozone (O₃) exposure are dependent on changes in iron homeostasis. After O₃ exposure, healthy volunteers demonstrated increased lavage concentrations of iron, transferrin, lactoferrin, and ferritin. In normal rats, alterations of iron metabolism after ozone were immediate and preceded the inflammatory influx. To test for participation of this disruption in iron homeostasis in lung injury following O₃ inhalation, Belgrade rats, deficient in functional divalent metal transporter-1 (DMT1) as a means of iron uptake, and controls were exposed to O₃. Belgrade rats had a larger disruption of iron homeostasis and greater injury than control rats. Human bronchial epithelial (HBE) cells exposed to O₃ demonstrated higher non-heme iron and ferritin concentrations relative to filtered air exposure. Aldehyde generation and IL-8 release by the HBE cells was also elevated following O₃ exposure. HEK293 cells with elevated expression of DMT1 were exposed to both filtered air and O₃. With exposure to O₃, elevated DMT1 expression diminished oxidative stress (i.e. aldehyde generation) and IL-8 release following ozone exposure. We conclude that iron participates critically in the oxidative stress and biological effects after ozone exposure.