Rats and guinea pigs were exposed to 0.8 mg ozone (O3)/m3 (approximately 0.4 ppm) for 12 hr during the daytime, 12 hr during the nighttime, or continuously to investigate circadian variation in O3-induced pulmonary toxicity during single and repeated O3 exposures. Biomarkers in bronchoalveolar lavage (BAL) fluid and lung tissues were measured as indicators of biochemical and inflammatory responses. Nighttime O3 exposure of rats resulted in larger increases of protein, albumin, and inflammatory cells in BAL fluid compared to those after daytime O3 exposure and this daytime-nighttime difference was statistically significant (p < 0.05). Single daytime or nighttime O3 exposure of guinea pigs resulted in comparable increases of BAL fluid proteins and inflammatory cells without a daytime-nighttime difference. Nighttime and continuous O3 exposure of rats for 3 days resulted in comparable increases in lung antioxidant enzyme activities, both of which differed statistically from effects from daytime O3 exposures (p < 0.05). Continuous O3 exposure of guinea pigs for 3 days caused, in general, statistically larger increases in lung tissue parameters compared to nighttime O3 exposures (p < 0.05). These results suggest that the extent of O3-induced acute pulmonary biochemical and inflammatory responses is directly related to the level of physical and respiratory activity. For rats, effects from continuous O3 exposure appear to be controlled by the nighttime, physically active period. In guinea pigs, the comparable responses following daytime or nighttime O3 exposure seem in accordance with their random behavioral daily activity pattern. This study supports the view that physical activity-related increases in inhaled dose significantly enhance the pulmonary O3 responses.