Tolerance to respiratory effects of O₃ has been demonstrated for anatomic and functional changes, but information about tolerance to O₃-induced airway hyperresponsiveness (AHR) is scarce. In guinea pigs exposed to air or O₃ (0.3 parts/million, 4 h/day, for 1, 3, 6, 12, 24, or 48 days, studied 16-18 h later), pulmonary insufflation pressure changes induced by intravenous substance P (SP, 0.032-3.2 µg/kg) were measured, then the animals were subjected to bronchoalveolar lavage (BAL). Bronchial rings with or without phosphoramidon were also evaluated 3 h after air or a single O₃ exposure. O₃ caused in vivo AHR (increased sensitivity) to SP after 1, 3, 6, 12, and 24 days of exposure compared with control. However, after 48 days of exposure, O₃ no longer caused AHR. Total cell, macrophage, neutrophil, and eosinophil counts in BAL were increased in most O₃-exposed groups. When data from all animals were pooled, we found a highly significant correlation between degree of airway responsiveness and total cells (r = 0.55), macrophages (r = 0.54), neutrophils (r = 0.47), and eosinophils (r = 0.53), suggesting that airway inflammation is involved in development of AHR to SP. Superoxide dismutase (SOD) levels in BAL fluids were increased (P < 0.05) after 1, 3, 6, and 12 days of O₃ exposure and returned to basal levels after 24 and 48 days of exposure. O₃ failed to induce hyperresponsiveness to SP in bronchial rings, and phosphoramidon increased responses to SP in air- and O₃-exposed groups, suggesting that neutral endopeptidase inactivation was not involved in O₃-induced AHR to SP in vivo. We conclude that chronic exposure to 0.3 ppm O₃, a concentration found in highly polluted cities, resulted in tolerance to AHR to SP in guinea pigs by an SOD-independent mechanism.