Ambient particulate matter (PM) concentrations have been associated with mortality and morbidity. Diesel exhaust particles (DEP) are present in ambient urban air PM. Coexisting with DEP (and PM) is ozone (O₃), which has the potential to react with some components of DEP. Some reports have shown increased lung injury in rats coexposed to PM and O₃, but it is unclear whether this increased injury was due to direct interaction between the pollutants or via other mechanisms. To examine whether O₃ can directly react with and affect PM bioactivity, we exposed DEP to O₃ in a cell-free in vitro system and then examined the bioactivity of the resultant DEP in a rat model of lung injury. Standard Reference Material 2975 (diesel exhaust PM) was initially exposed to 0.1 ppm O₃ for 48 h and then instilled intratracheally in Sprague–Dawley rats. Rat lung inflammation and injury was examined 24 h after instillation by lung lavage. The DEP exposed to 0.1 ppm O₃ was more potent in increasing neutrophilia, lavage total protein, and LDH activity compared to unexposed DEP. The increased DEP activity induced by the O₃ exposure was not attributable to alteration by air that was also present during the O₃ exposure. Exposure of DEP to a higher O₃ concentration (1.0 ppm) led to a decreased bioactivity of the particles. In contrast, carbon black particles, low in organic content relative to DEP, did not exhibit an increase in any of the bioactivities examined after exposure to 0.1 ppm O₃. DEP incorporated O₃ (labeled with ¹⁸O) in a linear fashion. These data suggest that ambient concentrations of O₃ can increase the biological potency of DEP. The ozonized DEP may play a role in the induction of lung responses by ambient PM.