We tested if there is a direct relationship between reactive oxygen species and citric acid-induced airway constriction. Guinea pigs were divided into two groups: control and dimethylthiourea (a hydroxyl radical scavenger). The animals in each group were further separated into four subgroups: baseline, recovery 2-3 min, recovery 10 min, and recovery 20 min. Each animal was anesthetized, cannulated, paralyzed, and artificially ventilated. Citric acid aerosol inhalation caused the following significant changes in the control group during the recovery period: airway constriction for at least 20 min, increases in luminol-amplified t-butyl hydroperoxide-initiated chemiluminescence counts in the bronchoalveolar lavage samples at 2-3 and 20 min, an increase in bronchoalveolar lavage fluid substance P level at 2-3 min, and elevations in the bronchoalveolar lavage fluid total cell and neutrophil numbers at 20 min. All citric acid-induced alterations were prevented by dimethylthiourea pretreatment. These results suggest that citric acid inhalation induces the initial release of reactive oxygen species and tachykinins, which causes further cellular infiltration and sustained airway constriction.