Exposure to traffic-related air pollution is associated with increased cardiovascular morbidity and mortality. Though the mechanisms between exposure and outcome are unclear, previous findings suggest that oxidative stress plays a role in more than one proposed pathway. Diesel exhaust, the dominant source of urban air pollution, comprises a mixture of components that, when inhaled, may directly or indirectly increase reactive oxygen species, decrease antioxidant capacity, and alter redox signaling. Based on the hypothesis that antioxidant levels reflect this response, we investigated the effect of diesel exhaust exposure on blood levels of the major cellular antioxidant glutathione in human subjects, and whether this response could be blunted by antioxidant pre-treatment. Seventeen healthy, nonsmoking adults, age 18-49, participated in a double-blind, crossover controlled exposure study. Subjects completed 120-minute exposures, randomized to order, to filtered air and 200 ug/m3 diesel exhaust, each with and without prior antioxidant treatment (ascorbate + N-acetylcysteine or placebo). Total and oxidized glutathione was measured in whole blood, drawn prior to and two hours after exposure. We further examined effect modification by genotype for the glutamate cysteine ligase catalytic subunit (GCLc). Compared with filtered air, the ratio of reduced to oxidized glutathione was significantly lower with exposure to diesel exhaust (-328; CI:-545, -111; p=0.003). Antioxidant treatment and genotype did not significantly modify this effect. Diesel exhaust inhalation was associated with a decrease in the ratio of reduced to oxidized glutathione in healthy adults, an effect that was not significantly attenuated by antioxidant treatment. This finding supports the theory that oxidative stress is one mechanism involved in the adverse effects associated with acute exposures to air pollution. Decreased antioxidant status may also exacerbate susceptibility to other oxidative insult.